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Novel septin 9 repeat motifs altered in neuralgic amyotrophy bind and bundle microtubules

Novel septin 9 repeat motifs interact with the acidic C-terminal tails of β-tubulin to promote microtubule bundling and asymmetric neurite growth.

Introduction
Septins are hetero-oligomeric GTP-binding proteins that assemble into nonpolar filamentous structures, which are essential for microtubule (MT)-dependent cell processes such as mitosis and vesicle transport (Spiliotis, 2010;Saarikangas and Barral, 2011;Mostowy and Cossart, 2012). In mammalian cells, septin depletion affects MT organization, dynamics, and post-translational modifications (Spiliotis et al., 2008;Bowen et al., 2011), but how septins interact with MTs is unknown. Septin 9 (SEPT9) is a ubiquitously expressed septin that caps the ends of septin heteromers (Füchtbauer et al., 2011;Kim et al., 2011;Sellin et al., 2011). Alternative splicing and translation start sites give rise to SEPT9 isoforms, which differ in the length and sequence of their N terminus, which is critical for the association of septin heteromers with MTs (Sellin et al., 2012). Missense mutations in the N terminus of the SEPT9 are genetically linked to hereditary neuralgic amyotrophy (HNA), a hereditary neuralgic disorder with symptoms (shoulder/arm pain and atrophy) similar to those of the idiopathic Parsonage-Turner syndrome (Kuhlenbäumer et al., 2005;Hannibal et al., 2009;van Alfen, 2011). Abnormal expression of SEPT9 isoforms has also been reported in many cancers (Connolly et al., 2011a). Overexpression of SEPT9 isoforms induces oncogenic phenotypes including resistance to the anti-cancer drug paclitaxel, which stabilizes and bundles MTs (Orr et al., 2003;Gonzalez et al., 2007;Chacko et al., 2012). Unraveling the mechanism of SEPT9 interaction with MTs could point to the molecular roles of SEPT9 in these disorders and lead to therapeutic strategies.

The basic N-terminal domain of SEPT9 binds and bundles MTs by interacting with the acidic C-terminal tails of -tubulin
To determine how SEPT9 interacts with MTs, we first sought to identify the SEPT9 domain that binds MTs using in vitro cosedimentation assays. Recombinant His-tagged fragments were made from the longest SEPT9_i1 isoform (SEPT9-FL; Fig. 1 A), which consists of the GTP-binding domain (aa 283-586; SEPT9-G) and a structurally disordered N-terminal tail (aa 1-283; SEPT9-N; Fig. S1 A) with a basic region (aa 1-142; SEPT9-B) and a S eptin 9 (SEPT9) interacts with microtubules (MTs) and is mutated in hereditary neuralgic amyotrophy (HNA), an autosomal-dominant neuropathy. The mechanism of SEPT9 interaction with MTs and the molecular basis of HNA are unknown. Here, we show that the N-terminal domain of SEPT9 contains the novel repeat motifs K/R-x-x-E/D and R/K-R-x-E, which bind and bundle MTs by interacting with the acidic C-terminal tails of -tubulin. Alanine scanning mutagenesis revealed that the K/R-R/x-x-E/D motifs pair electrostatically with one another and the tails of -tubulin, enabling septin-septin interactions that link MTs together. SEPT9 isoforms lacking repeat motifs or containing the HNA-linked mutation R88W, which maps to the R/K-R-x-E motif, diminished intracellular MT bundling and impaired asymmetric neurite growth in PC-12 cells. Thus, the SEPT9 repeat motifs bind and bundle MTs, and thereby promote asymmetric neurite growth. These results provide the first insight into the mechanism of septin interaction with MTs and the molecular and cellular basis of HNA.
Novel septin 9 repeat motifs altered in neuralgic amyotrophy bind and bundle microtubules scrambled or -tubulin CTT (Fig. 2, E and F). Interestingly, the CTTs of Iand III-tubulin isotypes did not have an effect, indicating that SEPT9 interacts preferentially with II-tubulin ( Fig. 2 G). Preferential interaction of SEPT9 with -tubulin was also confirmed with a blot-overlay assay, in which and -tubulin subunits were separated by SDS-PAGE (Fig. 2 H). We concluded that electrostatic interactions between the acidic CTT of -tubulin and the basic N-terminal domain of SEPT9 play a central role in SEPT9-MT binding and bundling. Novel SEPT9 repeat motifs K/R-x-x-E/D and R/K-R-x-E provide a mechanism for

MT bundling
To hone in on the mechanism of SEPT9-MT interaction, we sought to identify domains or repeat motifs similar to the positively charged MT-binding domains of MAPs, MT plus endtracking proteins and motors (Amos and Schlieper, 2005;Marx et al., 2006;Akhmanova and Steinmetz, 2008). Although we were not able to identify any known motifs, the N terminus of SEPT9_i1 contains multiple repeats of the tetrapeptide sequences K/R-x-x-E/D and R/K-R-x-E (Fig. 3, A and B), which are often flanked by proline or serine residues. By truncating the basic N-terminal domain of SEPT9_i1, we determined that aa 61-113 comprise the most minimal region of SEPT9_i1 that binds and bundles MTs (Fig. S2, A-C) and contains multiple repeats of the identified motifs.
We set out to elucidate the role of these novel motifs in MT binding and bundling by alanine scanning mutagenesis. The first half of the SEPT9_i1(61-113) peptide contains a region (R1) of three K/R-x-x-E/D motifs and the second half (R2) consists of a K-x-x-E motif flanked by two R-R-x-E motifs ( Fig. 3 C); neither R1 nor R2 alone bound or bundled MTs. Alanine substitution of the basic residues in two or all three of the R1 motifs resulted in an 50% decrease in MT binding (Fig. 3 D and Fig. S2 D). A similar decrease was observed by mutating the basic residues of the R2 motifs (Fig. 3 D and Fig. S2 E). We assessed the effects of the same mutations in MT bundling using a low speed sedimentation assay. Strikingly, alanine substitutions of the basic residues of the R-R-x-E motifs of R2 decreased MT bundling, but mutating the basic residues of the K/R-x-x-E/D motifs of R1 did not decrease MT bundling (Fig. 3 E; Fig. S2, F and G). These data indicate that although the basic residues of both K/R-x-x-E/D and R-R-x-E contribute to MT binding, the arginine residues of the R-R-x-E motifs are uniquely critical for MT bundling.
Next, we assessed the role of the acidic residues. Alanine substitutions of the acidic residues in one or two of the motifs of either R1 or R2 resulted in a moderate (30%) increase of MT-SEPT9_i1(61-113) binding ( Fig. 3 F and Fig. S2 H). MT bundling, however, decreased by 50% when the acidic residues in two of the K/R-x-x-E/D motifs of the R1 region were mutated ( These results show that although the acidic residues dampen somewhat the electrostatic affinity of all motifs for the CTTs of -tubulin, the acidic residues of the K/R-x-x-E/D motifs (R1) proline-rich acidic domain (aa 143-283; SEPT9-A). SEPT9-FL co-sedimented with MTs ( Fig. 1, B and J) and the apparent K d value was 3.2 µM (Fig. S1 B). In contrast to SEPT9-G and SEPT9-A, which pelleted with MTs weakly (Fig. 1, D-F and J), SEPT9-N and SEPT9-B bound MTs similarly to SEPT9-FL ( Fig. 1, C, E, and J).
Next, low speed MT pelleting and visual MT assays were used to examine whether SEPT9 can bundle MTs. At low speed (8,000 g), MT sedimentation was increased by SEPT9-FL, SEPT9-N, and SEPT9-B (Fig. 1, G-I and K), but SEPT9-G and SEPT9-A had no effect ( Fig. 1, H, I, and K). Incubation of fluorescent MTs with SEPT9-FL showed a marked increase in the length, thickness, and brightness of MT bundles (Fig. 1,L,N,and O). This bundling effect was independently confirmed by negative stain EM. MTs were arranged in bundles and doublets of parallel tubules that made tight contacts ( Fig. 1 M). Elongated MT bundles were also observed with fluorescent MTs that were stabilized with the nonhydrolyzable GTP analogue GMP-CPP ( Fig. S1 C). Decoration of paclitaxel-and GMP-CPP-stabilized MT bundles with recombinant GFPtagged SEPT9-FL, but not SEPT2, demonstrated that SEPT9 binds and bundles MTs strongly and specifically (Fig. S1, D-I).
In agreement with the MT sedimentation assays, significant bundling of fluorescent MTs was observed for only SEPT9-N and SEPT9-B (Fig. 1, L, N, and O). These data indicate that SEPT9 binds and bundles MTs via its basic N-terminal domain. Because SEPT9 forms hetero-oligomeric complexes with other septins (e.g., SEPT7, SEPT6), we purified SEPT9 in the presence of SEPT6/7 and tested how SEPT6/7/9 complexes affect MT bundling compared with SEPT9 alone or SEPT2/6/7. SEPT6/7/9 induced low speed sedimentation of MTs at a concentration (0.2 µM) that neither SEPT9 nor SEPT2/6/7 had an effect (Fig. S1, J and K). Thus, SEPT9 appears to be a key subunit for the bundling of MTs by septin complexes.
Electrostatic interactions between positively charged domains and the negatively charged C-terminal tails (CTTs) of tubulin underlie the mechanism by which some microtubuleassociated proteins (MAPs) and kinesin motors associate with MTs (Amos and Schlieper, 2005;Marx et al., 2006). We hypothesized that electrostatics could similarly be involved in SEPT9-MT binding and bundling. Consistent with this possibility, MT bundling by SEPT9 was attenuated with increasing ionic strength ( Fig. S1 L). We therefore tested whether the acidic CTTs of tubulin are required for MT-SEPT9 binding and aimed to identify the tubulin subunit(s) that associate with SEPT9.
We first repeated the MT-SEPT9 co-sedimentation and visual assays using pre-polymerized MTs treated with subtilisin, which removes the CTTs of tubulin; 50% of -tubulin and 90% -tubulin tails were cleaved. Pelleting of SEPT9_i1 with MTs decreased by >50% after subtilisin-treatment ( Fig. 2 A). Fluorescent MT elongation and bundling by SEPT9_i1 were also attenuated after subtilisin treatment (Fig. 2, B-D). To further test whether SEPT9-MT binding involves the CTTs of tubulin, we performed MT-SEPT9 pelleting assays in the presence of synthetic peptides, whose sequences corresponded to the CTTs of and II-tubulin and a scrambled control. MT-SEPT9 binding decreased in a dose-dependent manner by II, but not Novel SEPT9 repeat motifs bundle microtubules • Bai et al.  (H) Increasing amounts of bovine brain tubulin was separated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes, which were stained with Ponceau S red. Membranes were blotted with DM1A and TUB2.1 antibodies against -and -tubulin, respectively, and overlaid with His-tagged SEPT9-FL and SEPT2, which were detected with anti-His antibodies. Novel SEPT9 repeat motifs bundle microtubules • Bai et al.

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; Teng et al., 2001;Feltrin et al., 2012). We hypothesized that SEPT9 isoforms that lack K/R-R/x-x-E/D motifs from their N terminus could affect MT bundling and asymmetric neurite growth. Among the known SEPT9 protein isoforms, SEPT9_i4 lacks the N-terminal 164 aa and therefore most of the MT-binding and -bundling repeat motifs of SEPT9_i1. To test if the absence of these motifs affects intracellular MT bundling, we expressed GFP-tagged chimeras of these isoforms in MDCK cells, in which septins were previously shown to colocalize with MTs (Spiliotis et al., 2008;Bowen et al., 2011). We found that SEPT9_i1 colocalized strongly with perinuclear MT bundles, whereas SEPT9_i4 was distributed at peripheral lamellae showing little to no colocalization with MTs (Fig. 4, A and C). Upon treatment with paclitaxel, an MT-stabilizing drug that induces MT bundling, SEPT9_i1 colocalized with MT bundles extensively, whereas SEPT9_i4 showed little colocalization with MTs (Fig. 4, B and C). Interestingly, paclitaxel-treated cells that expressed SEPT9_i4 cells appeared to have less number of long and straight MT bundles (Fig. 4 B). This effect was quantified by measuring the relative percentage of MTs with fluorescence intensities 5× or 10× greater than the mean intensity of the are uniquely critical for MT bundling. Because the basic residues of the R-R-x-E motif are also uniquely involved in MT bundling, we posit that the extra arginine residues of the R-R-x-E motifs allow for additional interactions with the acidic residues of the K/R-x-x-E/D motifs, enabling homophilic trans interactions between SEPT9_i1(61-113) peptides that cross-link MTs into bundles (Fig. 3 H). Consistent with this model, a GST-tagged version of the N terminus of SEPT9 bound His-tagged SEPT9-N and SEPT9-FL (Fig. S2, J and K). Thus, homophilic trans interactions between the N termini of SEPT9 are likely to facilitate septin and MT cross-linking. Given the mechanistic role of the K/R-R/x-x-E/D motifs in MT binding and bundling in vitro, we examined the functional significance of these motifs for intracellular MT bundling and neuronal morphogenesis, which requires MT bundling by MAPs (e.g., tau, MAP1B) that support the asymmetric growth of neuronal protrusions termed neurites (Caceres and Kosik, , and percentage of total tubulin pelleted at 8,000 g (E) in three independent experiments. Representative gels are shown in Fig. S2. (F and G) Graphs show percentage of total 61-113 peptide (wild-type and acidic residue mutants) pelleted with MTs at 39,000 g (F), and percentage of total tubulin pelleted at 8,000 g (G) in three independent experiments. Representative gels are shown in Fig. S2. (H) Schematic shows a model of electrostatic interactions between the acidic (red) CTTs of tubulin and the basic (blue) residues of the SEPT9 repeat motifs. MT cross-linking is achieved by interactions between the acidic and basic residues of the K/R-x-x-E/D and R/K-R-x-E motifs, respectively. the percentage of cells with a single neurite by 50% relative to GFP-expressing cells (Fig. 5 I). The R88W mutant, however, increased the overall percentage of cells with more than three neurites (Fig. 5 I). Taken together, these data show that the HNA-linked R88W mutation has adverse effects on MT bundling and asymmetric neurite growth.
Our results provide the first insight into the mechanism of septin interaction with MTs. We have shown that the N terminus of SEPT9 binds and bundles MTs via repeat motifs similar to those of conventional MAPs (Noble et al., 1989;Butner and Kirschner, 1991;Cravchik et al., 1994;Ferralli et al., 1994). Formation of uniform linear arrays of bundled MTs is likely to involve highly ordered cis and trans interactions between SEPT9 containing hetero-oligomers. Cis interactions between the GTPbinding domains of septins mediate their assembly into heteromeric filaments (Sirajuddin et al., 2007). Conversely, trans interactions between the C-terminal coiled-coil domains of septins mediate the pairing of septin filaments (John et al., 2007;Bertin et al., 2008;DeMay et al., 2011;de Almeida Marques et al., 2012). SEPT9 molecules lack C-terminal tails, but our data indicate that their N-terminal extensions could mediate trans interactions by electrostatic pairing between the acidic and basic residues of the K/R-x-x-E/D and R/K-R-x-E motifs, respectively. SEPT9-mediated MT cross-linking could be further reinforced by the trans interactions between the C-terminal coiled-coil domains of SEPT2, SEPT6, and SEPT7, which hetero-oligomerize with SEPT9 (Kim et al., 2011;Sellin et al., 2011). Although SEPT2 does not bind MTs (Fig. S1), previous work indicates that SEPT7 and possibly SEPT6 interact with MTs (Hu et al., 2012;Moon et al., 2013). These subunits, however, lack the repeat motifs of SEPT9.
Septins colocalize with MTs in various cell types, but the physiological significance of this interaction is poorly understood. Here, we have found that SEPT9 and its N-terminal repeat motifs affect intracellular MT bundling and asymmetric neurite growth. Importantly, expression of SEPT9 isoforms with deletions (SEPT9_i4) and mutations (HNA-linked R88W) in the repeated K/R-R/x-x-E/D sequence motifs impair MT bundling and asymmetric neurite growth. Interestingly, cancer cells that overexpress SEPT9_i4 have increased resistance to the drug paclitaxel (Chacko et al., 2012). It is unknown if paclitaxelmediated MT bundling contributes to the cytotoxicity of the drug, but our data indicate that SEPT9 and its repeat motifs are partly required for paclitaxel-induced MT bundling. This role of SEPT9, however, could vary between different cell types. Given the preferential interaction of SEPT9 with II-tubulin, variations in the expression of -tubulin isotypes could affect septin association with MTs. In summary, our results suggest that alterations in SEPT9 isoform expression trigger changes in the intracellular organization and function of MTs, and thereby could contribute to the pathology of HNA and cancer.

Materials and methods
Cells, peptides, and plasmids MDCKII/G cells were maintained in low glucose DME media (Sigma-Aldrich) supplemented with 10% FBS (Cell Generation) and 1 g/liter NaHCO 3 . MDCK cells were transfected with the plasmids pEGFP-SEPT9_v1 dimmest single peripheral MTs, using a segmentation analysis of 3D confocal image stacks as previously published (Sammak and Borisy, 1988;Bowen et al., 2011). MDCK cells that expressed SEPT9_i4-GFP had consistently lower percentage of 5× or 10× MTs compared with control and SEPT9_i1-GFP in the presence or absence of paclitaxel (Fig. 4, D and E). A lower percentage of MT bundles was also observed in fibroblasts from Sept9 knockout mice at steady-state and after treatment with paclitaxel (Fig. S3). Thus, SEPT9 is critical for the intracellular organization of MTs into bundles.
During neuronal morphogenesis, MT bundling supports the asymmetric growth of a neurite, which extends faster and longer than other neurites to become the presumptive axon. To test whether SEPT9_i1 and the K/R-R/x-x-E/D motifs affect neurite growth, we assayed for NGF-induced neurite formation in PC12 cells. After 48 and 72 h of NGF treatment, the percentage of GFP-SEPT9_i1-expressing cells with single neurites was respectively fivefold and twofold higher than control cells (Fig. 4, F and G). In contrast to this amplification of asymmetric neurite growth by SEPT9_i1, expression of SEPT9_i4 had the opposite effect, increasing the percentage of cells with more than three neurites by twofold after 48 h of NGF treatment (Fig. 4, F and G). Notably, after 72 h of NGF exposure, the percentage of SEPT9_i4expressing cells with single neurites was twofold lower than control cells (Fig. 4 G) and the average neurite length was shorter than the length of single neurites in SEPT9_i1-expressing cells (2.3 vs. 3.3 times the cell width). These results indicate that the MT-bundling properties of SEPT9 promote asymmetric neurite growth, which depends on the presence of K/R-R/x-x-E/D motifs within the N-terminal sequence of SEPT9.

HNA-linked mutation R88W impairs MT bundling and asymmetric neurite growth
Alterations in the N-terminal sequence of SEPT9 have been genetically linked to HNA, a neuralgic disorder whose molecular etiology remains unknown (Kuhlenbäumer et al., 2005;Hannibal et al., 2009;van Alfen, 2011). Several HNA patients carry the mutation R88W in isoform 3 of SEPT9 (Hannibal et al., 2009), which has one K/R-x-x-E/D motif less than SEPT9_i1 and contains seven unique residues in place of aa 1-25 of SEPT9_i1. Because the R88W mutation of SEPT9_i3 maps to the first arginine of an R-R-x-E motif, we asked whether this mutation affects the MT-binding and -bundling properties of SEPT9_i3. The missense mutation did not affect high speed pelleting of SEPT9_i3 with MTs (Fig. 5, A and C), but low speed sedimentation of MTs decreased by 30% (Fig. 5, B and D). In fluorescent MT-bundling assays, the R88W mutation weakened the MT-bundling effects of SEPT9_i3 (Fig. 5, E-G). To determine if the R88W mutation alters MT bundling in vivo, we obtained dermal fibroblasts from healthy control individuals and HNA patients with the R88W genotype. We found that the fraction of MTs with 5× the mean intensity of single MTs was lower in the HNA patient fibroblasts compared with cells from a healthy individual (Fig. 5 H). Given the role of SEPT9_i1 and its repeat motifs in asymmetric neurite growth, we tested how the wildtype and R88W mutant versions of SEPT9_i3 affect neurite outgrowth in PC12 cells. Expression of GFP-SEPT9_i3 increased Industries) was used to take a skin sample from the extensor surface of the upper arm. After removal of the subcutaneous fatty tissue, skin explants were cultured in high glucose DME media with 20% FBS for 5-7 d. Skin fibroblasts that grew out of the explants were isolated and passaged. Experiments with R88W and control 1 fibroblasts were performed at passages 7-11 and 9-13, respectively. The peptides NH 2 -CEVGVDSVEGEGEEE-GEEY-COOH (-tubulin CTT), NH 2 -CQETAEEYQDEEQGEADAEDFG-COOH (control, scrambled II-tubulin CTT), and NH 2 -CQYQDATAEEEEDFGE-EAEEEA-COOH (I tubulin CTT) were purchased at >95% purity from GenScript. The peptides NH 2 -CQYQDATADEQGEFEEEEGEDEA-COOH (II-tubulin CTT) and NH 2 -CQYQDATAEEEGEMYEDDEEESEAQGPK-COOH (III-tubulin CTT) were purchased at >95% purity from Life-Tein, LLC.
His-SEPT9_i1-expressing plasmid was constructed by PCR amplifying human SEPT9_i1 (NP_001106963) using the primers 5-CGTAAGCTTG-CATGAAGAAGTCTTACTC-3 and 5-GTACTCGAGCTACATCTCTGG-GGC-3 and cloning the amplified fragment into the HindIII and XhoI sites of and pEGFP-SEPT9_v4, which encode respectively for GFP-tagged SEPT9_i1 and SEPT9_i4 and were constructed by PCR amplification of SEPT9_v1 (AF189713) and SEPT9_v4 (AJ312322) from normal breast tissue cDNA and insertion into pEGFP-C2 (Connolly et al., 2011b). PC-12 cells were maintained in high glucose DME supplemented with 6% donor-defined equine serum (Hyclone), 6% defined bovine calf serum (Hyclone), and 1 g/liter NaHCO 3 . PC-12 differentiation and neurite growth were induced after 24 h of transfection by incubation in low serum (1% horse serum, 1% bovine calf serum) DME containing 100 ng/ml 2.5S NGF (Harlan Biosciences). Embryonic fibroblasts derived from Sept9 cond/cond and Sept9 del/del mice were provided by E.-M. Füchtbauer (University of Aarhus, Aarhus, Denmark) and maintained in DME with 10% FBS as described previously (Füchtbauer et al., 2011). Dermal cells were derived from skin biopsies taken from a healthy individual (control 1) and an HNA patient with the SEPT9_i3_R88W mutation (c.262C>T). Both subjects provided written informed consents and the biopsy protocol was approved by the University of Kiel ethics committee. Under local anesthesia, a standard skin-biopsy punch (diameter 3 mm; Kai Figure 5. HNA-linked mutation R88W impairs MT bundling and neurite asymmetry. (A and B) Gels show supernatant (S) and pellet (P) fractions from high (A; 39,000 g) and low (B; 8,000 g) speed MT-pelleting assays. (C and D) Graphs show percentages of total protein co-pelleted with MTs (C) and percentage of total tubulin pelleted (D) in three independent experiments. (E) Images show X-rhodamine-labeled MTs after mixing with recombinant SEPT9_i3 and SEPT9_i3(R88W). Bars, 10 µm. (F and G) Plots show the intensity of X-rhodamine fluorescence per micron of MT (F; n = 100) and the length of MTs (G; n = 100) per condition. (H) Images of dermal cells from healthy individual (control 1) and HNA patient with the R88W genotype stained for -tubulin. Bars, 10 µm. Plot shows intensity of putative MT bundles with 5× the mean intensity of single MTs as percentage of total MT intensity (n = 15). (I) Phase-contrast images show PC12 cells transfected with GFP and GFP-tagged wild-type and R88W SEPT9_i3. Insets show GFP fluorescence in inverted monochrome. Graph shows percentage of cells (n = 90) with one or more neurites. Pooled data from three independent experiments are shown. Bars, 10 µm.
High and low speed MT cosedimentation and protein binding assays Bovine brain tubulin (10 µM; >99% pure; Cytoskeleton, Inc.) was polymerized in G-PEM (80 mM Pipes, pH 6.9, 1 mM EGTA, 1 mM MgCl 2 , 1 mM GTP, and 10% glycerol) plus 80 µM paclitaxel for 30 min at 37°C. Subtilisin-cleaved MTs (S-MTs) were prepared by incubating pre-polymerized MTs with 30 µg/ml subtilisin (Sigma-Aldrich) at 37°C for 3 h. The reaction was terminated by addition of 2 mM PMSF. S-MTs were pelleted at 16,000 g on a cushion buffer (80 mM K-Pipes, pH 6.9, 1 mM MgCl 2 , 1 mM EGTA, 60% glycerol, and 20 µM paclitaxel) for 10 min at room temperature and then resuspended in 20 µl G-PEM with 20 µM paclitaxel. S-MTs were obtained from the supernatant fraction after re-centrifugation on 40 µl of cushion buffer at 12,000 g for 5 min. In high speed sedimentation assays, untreated, mock-, or subtilisin-treated MTs were incubated with recombinant SEPT9 proteins (10 µM) or SEPT9(61-113) peptides (30 µM) for 20 min at room temperature. Each reaction was placed on cushion buffer and centrifuged at 39,000 g (high speed) for 20 min at 25°C in an ultracentrifuge (Optima TL100; Beckman Coulter). In low speed sedimentation assays, MTs were incubated with recombinant SEPT9 proteins (2 µM), SEPT9(61-113) peptides (30 µM), or SEPT2/6/7, SEPT6/7/9_i1 complexes and SEPT9_i1 (0.2 µM; Fig. S1, J and K) for 10 min at room temperature. Each reaction was placed on cushion buffer and centrifuged at 8,000 g for 5 min. Pellets were resuspended in PBS of the same volume with the supernatants. Equal volumes of pellet and supernatant were loaded onto 10% SDS-PAGE and gels were stained with Coomassie Brilliant Blue. Gels were scanned and protein band densities were quantified with the Odyssey infrared scanning system (LI-COR Biosciences).
Binding assays between GST-and His-tagged proteins were performed (Fig. S2, J and K) by incubating 25 µg of purified GST-SEPT9-N or GST-SEPT9-G with 20 µl glutathione agarose 4B beads for 15 min. After washing three times with GST binding buffer, beads were incubated with 20 µg His-SEPT2, His-SEPT9, His-SEPT9-N, or His-SEPT9-G for 1.5 h. Beads were washed with GST binding buffer five times before resuspended with loading buffer and boiled. Samples were loaded into 10% SDS-PAGE gels and stained with Coomassie Brilliant Blue.
Visualization of MT bundling by fluorescence and electron microscopy X-rhodamine-labeled bovine brain tubulin (10 µM; Cytoskeleton, Inc.) was polymerized in G-PEM buffer containing 80 µM paclitaxel. MTs were incubated with recombinant proteins (20 nM) in G-PEM buffer containing 20 µM paclitaxel for 10 min at room temperature. An aliquot (5 µl) of each reaction mix was mounted on a slide and sealed with a glass coverslip and nail polish. Slides were imaged on a fluorescent microscope (Axio Observer; Carl Zeiss) equipped with a Plan-Apo 63×/1.40 NA objective, a deep-cooled CCD camera (ORCA-AG; Hamamatsu Photonics), and Slidebook 5.0 software (Intelligent Imaging Innovations). All images were taken in the TRITC channel at 50-ms exposures. MT lengths and intensities were quantified by masking individual MTs (>5 µm). MT length and average fluorescence intensity per pixel after background subtraction were measured using Slidebook 5.0 software.
For visualization of MT bundling by negative stain electron microscopy, pre-polymerized MTs (Cytoskeleton, Inc.) were mixed with recombinant SEPT9-FL and incubated with glow-discharged 300 mesh Holey carbon copper grids covered with a thin layer of carbon. Grids were washed with 2% uranyl formate and subsequently blotted and air dried. Images were collected with a transmission electron microscope (Tecnai T12; FEI) equipped with a CCD camera (UltraScan 4000; Gatan, Inc.) operating at accelerating voltage of 120 kV, defocus value of 950 nm, and magnification of 52,000.
For the bacterial expression of septin heterotrimers, the bicistronic plasmid SEPT6-H-SEPT7 was constructed for the simultaneous expression of nontagged (NT) SEPT6 and His-tagged SEPT7. First, His-SEPT7 expressing plasmid was constructed by PCR amplifying the rat SEPT7 transcript variant 2 (NM 0011137.4) using the primer 5-TCGGGATCCATGTCGGTCAGT-GCG-3 and 5-GTAAAGCTTTTAAAAGATCTTGCC-3 and cloning the amplified fragment into the BamHI and HindIII sites of pET-28a(+). Second, a His-tagged SEPT6 was constructed by PCR amplifying the human SEPT6_v3 (NP_665798) using the primer 5-TCGGGATCCATGGCAGCGACC-GATATAG-3 and 5-TCGAAGCTTTTAATTTTTCTTCTC-3 and the amplified fragment was inserted into the BamHI and HindIII sites of pET-28a(+). Using the QuikChange II Site-Directed Mutagenesis kit, this plasmid was modified by removing the His tag in front of SEPT6 and inserting a SpeI site in front of the stop codon of SEPT6. Subsequently, the SEPT7 gene and the upstream ribosome-binding site were excised from pET-His-SEPT7 by digesting with XbaI and HindIII, and the fragment was subcloned into the SpeI and HindIII sites of the modified pET-SEPT6 to create a SEPT6-H-SEPT7-expressing plasmid. The plasmids encoding for NT-SEPT2 and NT-SEPT9_i1 were made from pET plasmids expressing His-SEPT9 (see above) and His-SEPT2, which was constructed by PCR amplifying mouse SEPT2 (NP_006146) using the primer 5-TCGGGATCCATGTCTAAGCAA-CAACC-3 and 5-ATCCTCGAGTCACACATGCTGCCCG-3 and cloning into the BamHI and XhoI sites of pET-28a(+). The His-SEPT9 and His-SEPT2 sequences and their upstream ribosome binding sites were then excised using XbaI and XhoI, and subcloned into pET-15b, which contains an ampicillin resistance marker. His tag sequences were then truncated with the QuikChange II Site-Directed Mutagenesis kit to create NT-SEPT2-and NT-SEPT9_i1-expressing plasmid.


extfieldsofstudy: Biology Medicine

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Heat Shock Transcription Factor 1-Deficiency Attenuates Overloading-Associated Hypertrophy of Mouse Soleus Muscle

Hypertrophic stimuli, such as mechanical stress and overloading, induce stress response, which is mediated by heat shock transcription factor 1 (HSF1), and up-regulate heat shock proteins (HSPs) in mammalian skeletal muscles. Therefore, HSF1-associated stress response may play a key role in loading-associated skeletal muscle hypertrophy. The purpose of this study was to investigate the effects of HSF1-deficiency on skeletal muscle hypertrophy caused by overloading. Functional overloading on the left soleus was performed by cutting the distal tendons of gastrocnemius and plantaris muscles for 4 weeks. The right muscle served as the control. Soleus muscles from both hindlimbs were dissected 2 and 4 weeks after the operation. Hypertrophy of soleus muscle in HSF1-null mice was partially inhibited, compared with that in wild-type (C57BL/6J) mice. Absence of HSF1 partially attenuated the increase of muscle wet weight and fiber cross-sectional area of overloaded soleus muscle. Population of Pax7-positive muscle satellite cells in HSF1-null mice was significantly less than that in wild-type mice following 2 weeks of overloading (p<0.05). Significant up-regulations of interleukin (IL)-1β and tumor necrosis factor mRNAs were observed in HSF1-null, but not in wild-type, mice following 2 weeks of overloading. Overloading-related increases of IL-6 and AFT3 mRNA expressions seen after 2 weeks of overloading tended to decrease after 4 weeks in both types of mice. In HSF1-null mice, however, the significant overloading-related increase in the expression of IL-6, not ATF3, mRNA was noted even at 4th week. Inhibition of muscle hypertrophy might be attributed to the greater and prolonged enhancement of IL-6 expression. HSF1 and/or HSF1-mediated stress response may, in part, play a key role in loading-induced skeletal muscle hypertrophy.

Introduction
Mechanical loading is one of the important factors in the regulation of skeletal muscle size. Skeletal muscle is highly plastic and adapts to physical demand. Increase in the mechanical load on skeletal muscle causes hypertrophy, whereas unloading induces atrophy. It has been generally accepted that increased loading activates muscle satellite cells, which are skeletal muscle-specific stem cells, and stimulates muscle protein synthesis. Hypertrophied muscle fibers, caused by mechanical loading, have a larger diameter, greater protein content, and increased number of myonuclei compared with sedentary control fibers. Although loading-dependent muscle hypertrophy is attributed to both the activation of muscle satellite cells and the stimulation of protein synthesis [1,2], the mechanisms responsible for these systems are not fully elucidated.
Stress proteins, so-called heat shock proteins (HSPs), are upregulated by hypertrophic stimuli, such as mechanical stretch, loading, and heat stress [3,4]. HSPs, which act as the molecular chaperones, play a part of the tightly regulated systems for maintenance of cellular homeostasis for survival in response to various pathological conditions [5,6]. HSPs, especially inducible 70 kDa HSP (HSP70, so-called HSP72), as well as HSP25, are induced and protect against cellular stresses via so-called stress response [3,4,[7][8][9]. Since HSP25 and HSP72 function as important molecular chaperones [3,4,8], there are many reports showing the prevention of disuse-and/or immobilization-associated skeletal muscle atrophy by overexpression of HSP25 or HSP72 [10,11]. Previous studies also demonstrated up-regulations of HSP25, HSP72 and/or HSP90 in hypertrophied skeletal muscles of rats [12][13][14][15] and mice [13]. However, physiological role(s) of up-regulation of HSPs for induction of hypertrophy of skeletal muscle cells remains unclear.
Since HSP25 and HSP72 are up-regulated during regrowth of mouse soleus muscle from unloading-related atrophy [16], it is suggested that these proteins may play role(s) in overloadingassociated skeletal muscle hypertrophy. HSP47, which is known as a collagen specific HSP [17], in anti-gravitational rat soleus muscle is up-regulated by hypergravity [18]. However, it is still unknown whether HSF1-deficiency influences the expression levels of HSP25, HSP47, HSP72, and HSP90 during skeletal muscle hypertrophy.
Heat shock transcription factors (HSFs), which mediate stress response, up-regulate the expression of HSPs via binding to heat shock element, located at the up-stream region of HSP genes [19,20]. Among three HSFs (HSF1, HSF2, and HSF4) in mammals, HSF1 plays a crucial role in inducing HSPs, conferring cytoprotection against various stresses [21,22]. In the previous study, we observed that HSF1-deficiency retarded the regrowth of atrophied solues muscles, which are induced by hindlimb unloading [16]. Recently, we have also reported that HSF1deficiency did not affect the population level of Pax7-positive satellite cells in the untreated control muscle [23]. However, effects of HSF1-deficiency on the loading-associated skeletal muscle hypertrophy are still unknown.
HSF1 suppresses inflammatory genes, including interleukin-6 (IL-6), through activating transcription factor 3 (ATF3) [24]. On the contrary, it has been suggested that HSF1 and HSF2 activate IL-6 synthesis [25]. IL-6, which is well-known as a pleiotropic cytokine, associated with the regulation and coordination of immune response [26], and other pro-inflammatory cytokines, such as IL-1b and tumor necrosis factor (TNF), are potentially mitogenic for myoblasts, as well as inhibitors of myogenic differentiation [27][28][29]. Intracellular signaling of TNFa, one of TNF superfamily, impairs insulin/insulin-like growth factor signaling by insulin receptor substrate-1 and Akt (so-called protein kinase B: PKB) [30,31], which plays a key role in skeletal muscle hypertrophy [32]. These observations suggest that HSF1 may modulate skeletal muscle hypertrophy via the behavior of IL-6mediated muscle satellite cells. However, the effects of HSF1deficiency on the responses of pro-inflammatory cytokines, as well as Akt, during overloading-associated skeletal muscle hypertrophy are unclear. Therefore, the current study was performed to investigate the physiological role of HSF1 gene on overloadingassociated skeletal muscle hypertrophy by using the HSF1-null mice.

Animals
All animal protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Bethesda, MD) and were approved by the Animal Use Committee at Toyohashi SOZO University (2007001). All treatments for animals were performed under anesthesia with i.p. injection of sodium pentobarbital, and all efforts were made to prevent discomfort and suffering. Male HSF1-null and wild-type (ICR) mice were prepared as described previously [16]. Mice with 10-15 wk of age were used in this experiment (n = 12 in each type of mice). Two or three mice were housed in a cage (20631 cm and 13.5 cm height). All mice were housed in a vivarium room with 12:12-h light:dark cycle and 23uC and ,50% temperature and humidity, respectively. Solid food and water were provided ad libitum.

Functional overloading
Functional overloading on the left soleus was unilaterally performed by removal of the distal tendons of both plantaris and gastrocnemius muscles under anesthesia with intrapenitoneal injection of pentobarbital sodium as described earlier [2]. The contralateral right muscle received sham operation as the control. The muscle was kept intact, although it was exposed surgically. The skin was sutured and mice were housed in the cages. It has been reported that unilateral functional overloading does not affect the myosin heavy chain (MHC) gene expression in the contralateral muscle, and the locomortion and mobility were unaltered by the unilateral functional overloading within 1 day post-surgery [12].

Sampling
Soleus muscles were dissected from the both hindlimbs 2 and 4 weeks after the surgery. All muscles were rapidly weighed (wet weight) and divided into three portions cross-sectionally. Then, muscle sections were frozen in liquid nitrogen and stored at 280uC until analyzed.

Muscle protein content
The middle portions of muscles were homogenized in ,0.4 ml (0.1 ml/mg muscle wet wt) of tissue lysis reagent (CelLytic-MT, Sigma-Aldrich, St. Louis, MO) and completely solubilized by alkaline treatment with 2 N NaOH at 37uC for 1 h [16]. Protein concentration of the tissue lysate was determined by using protein assay kit (Bio-Rad, Hercules, CA) and bovine serum albumin (Sigma) as the standard. Total protein content in whole muscle was then calculated.

Real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR)
In the present study, the expressions of HSP mRNAs, including HSP25, HSP47, the constitutive cytosolic HSC70, so-called HSP73, stress inducible HSP72, and HSP90a, and the expressions of HSF mRNAs, including HSF1, HSF2, and HSF4, were assessed by real-time RT-PCR. Pro-inflammatory cytokine mRNAs, such as IL-6, ATF3, IL-1b, and TNF, were also evaluated. Total RNA was extracted from the proximal portion of muscle using the miRNeasy Mini kit (Qiagen, Hiden, Germany) according to the manufacturer's protocol. Samples (,40 ng of RNA) were reversetranscribed using the first strand cDNA synthesis kit according to the manufacturer's instructions {PrimeScript RT Master Mix (Perfect Real Time) for mRNA, Takara Bio, Otsu, Japan}. Synthesized cDNA was applied to real-time reverse transcription-PCR (Thermal Cycler Dice Real Time System II MRQ, Takara Bio) using Takara SYBR Premix Ex Taq II for mRNA, and analyzed using Takara Thermal Cycler Dice Real Time System Software (version 4.00) according to the manufacturer's instructions. The real-time cycle conditions were 95uC for 30 s followed by 40 cycles at 95uC for 5 s and at 60uC for 30 s for mRNA. Specificity was confirmed by electrophoretic analysis of the reaction products and by inclusion of template-or reverse transcriptase-free controls. To normalize the amount of total RNA present in each reaction, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard.

Histochemical and immunohistochemical analyses
Serial transverse cryosections (7-mm thick) of frozen distal portion of soleus muscles were cut at 220uC and mounted on the slide glasses. The sections were air-dried and stained to analyze the degree of muscle damage and repair and the cross-sectional area (CSA) of muscle fibers by staining using hematoxylin and eosin (H&E), and the profiles of Pax7-positive nuclei by the standard immunohistochemical technique, respectively [33].
Monoclonal anti-Pax7 antibody (Developmental Studies Hybridoma Bank, Iowa, IA, USA) was used for the detection of muscle satellite cells [34]. Cross sections were fixed with paraformaldehyde (4%), and then were post-fixed in ice-cold methanol. After blocking by using a reagent (1% Roche Blocking Regent; Roche Diagonost, Penzberg, Germany), samples were incubated with the primary antibodies for Pax7 and rabbit polyclonal anti-laminin. Sections were also incubated with the second primary antibodies for Cy3-conjugated antimouse IgG1 (diluted 1:500; Jackson Immuno Research, West Grove, PA, USA) and for fluorescein isothiocyanate-conjugated anti-rabbit IgG (diluted 1:500; Sigma). Nuclei were then stained for 15 min in a solution of 4'6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.5 mg/mL; Sigma). The images of muscle sections were incorporated into a personal computer (DP Manager version 2.2.1.195, Olympus Japan, Tokyo) by using a microscope (IX 81 Olympus Japan). The CSAs of ,100 fibers from each muscle were analyzed on laminin-stained images by using ImageJ.

Immunoblotting analyses
Expressions of HSP25, HSP47, HSC70, HSP72, HSP90a, and total and phosphorylated Akt proteins were assessed by immunoblotting assay. Proximal portions of the right muscles were homogenized in an isolation buffer of tissue lysis reagent (CelLytic-MT, Sigma-Aldrich) with 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1g/ml leupeptin with glass homogenizer. The homogenates were then sonicated and centrifuged at 12,000 rpm (4uC for 10 min), and the supernatant was collected. A part of the supernatant was solubilized in sodium-dodecylsulfate (SDS) sample buffer {30% (vol/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol, 2.3% (wt/vol) SDS, 62.5 mM Tris?HCl, 0.05% (wt/vol) bromophenol blue, and pH 6.8} at a concentration of 0.5 mg of protein per milliliter and boiled for 3 min. The SDS-polyacrylamide gel electrophoresis (PAGE) was carried out on 10 or 12.5% polyacrylamide {bisacrylamide/ acrylamide, 1:20 (wt/wt)} slab gel (6068561 mm) containing 0.5% SDS at a constant current of 20 mA for 120 min, as was reported previously [35]. Equal amounts of protein (20 mg) were loaded on each gel. Molecular weight markers (ECL DualVue Western Blotting Markers, GE Healthcare, Buckinghamshire, UK) were applied to both sides of 14 lanes as the internal controls for transfer process or electrophoresis.
Following SDS-PAGE, proteins were transferred to PVDF membranes (0.2 mm pore size, Bio-Rad) at a constant voltage of 100 V for 60 min at 4uC. The membranes were blocked for 1 h using a blocking buffer: 5% skim milk with 0.1% Tween 20 in Tris-buffered saline (TTBS) with pH 7.5. The membranes were incubated for 1 h with a polyclonal antibody for HSP25 (SPA-801, StressGen, Victoria, BC), HSC70 (SPA-816, StressGen), HSP72 (SPA-812, StressGen), p-Akt (9271, Cell Signaling Technology, Beverly, Mass., USA), total Akt (9272, Cell Signaling Technology), and then reacted with a secondary antibody (goat anti-rabbit immunoglobulinG conjugate to horseradish peroxidase, Cell Signaling Technology) for 1 or 2 h. To detect HSP47 and HSP90a, antiserum for HSP47 and HSP90a were generated by immunizing rabbit, as described previously [36,37]. After the final wash, protein bands were visualized using chemiluminescence (ECL Advance Western blotting kit, GE Healthcare), and signal density was measured using Light-Capture (AE-6971) with CS Analyzer version 2.08b (ATTO, Tokyo, Japan). The activation level of Akt was evaluated by using the phosphorylated level of Akt relative to the total expression form (p-Akt/t-Akt), since phosphorylated form of Akt is enzymatically active [38,39]. Each sample was analyzed in duplicate, at least, to ensure that results were not influenced by loading errors. The GAPDH (G9545, Sigma-Aldrich) was evaluated to ensure the equal loading. Standard curves were constructed during the preliminary experiments to ensure linearity.

Statistical analyses
All values were expressed as means 6 SEMs. Statistical significances for body and muscle weights, protein content, expression levels of mRNA and protein were tested by using 2way (mice and time for body weight; mice and treatments for other measurements except HSF1 mRNA) analysis of variance (AN-OVA). When any significant main effects (factors) and interactions among factors were observed, Turkey-Kramer post hoc test was performed in each effect or among groups. For HSF1 mRNA, data was analyzed by using one-way ANOVA followed by Turkey-Kramer post hoc test. The significance level was accepted at p,0.05.

Body weight and soleus muscle wet weight
Changes in the body and muscle weights and muscle protein contents of HSF1-null and wild-type mice are shown in Figure 1. Although the body weight in each type of mice was identical between the 2nd and 4th week, that of wild-type mice was constantly higher than that in HSF1-null mice (p,0.05). There was no significant difference in the absolute muscle wet weight and proteins content of the contralateral control soleus muscle between wild-type and HSF-null mice. In wild-type mice, both muscle wet weight and protein content were significantly increased by 2 weeks of overloading, compared with the values of contralateral control (p,0.05). On the other hand, there was no significant effect of 2week overloading on muscle weight and protein content in HSF1null mice. Significant increase in wet weight and protein content in overloaded muscle was observed in wild-type and HSF1-null mice following 4 weeks of overloading (p,0.05). Increment of wet weight and protein content in overloaded soleus muscle relative to the contralateral control was significantly higher in wild-type mice than that in HSF1-null mice (Fig. 2, p,0.05). Figure 3A shows the representative cross-sectional images of soleus muscle in response to 2 and 4 weeks of overloading. Although there were no regenerating and damaged fibers in control and overloaded soleus muscle of both wild-type and HSF1null mice, muscle fiber hypertrophy was noted in overloaded muscles.

Fiber CSA
Responses of mean fiber CSAs during the experimental period are shown in Figure 3B. There was no significant difference in fiber CSA of control soleus muscle between wild-type and HSF1null mice. Mean fiber CSAs in wild-type mice were significantly increased by 2 and 4 weeks of overloading (p,0.05). Even though a significant increase in fiber CSA was observed following 4 weeks of overloading in HSF1-null mice (p,0.05), significant hypertrophy was not induced following 2 weeks of overloading. Figure 4 shows the changes in the distribution of Pax7-positive nuclei relative to total myonuclei in response to 2 and 4 weeks of overloading. There was no significant difference in the population of Pax7-positive nuclei of the contralateral control muscle between wild-type and HSF1-null mice. The population of Pax7-postive nuclei in wild-type mice was significantly increased after 2 weeks of overloading (p,0.05), but not in HSF1-null mice. The population of Pax7-positive nuclei in the overloaded muscle in wild-type mice was greater than that in HSF1-null mice (p,0.05). Following 4 weeks of overloading, insignificantly higher level of population of Pax7-positive nuclei was observed in both types of mice, compared with the level of control (p.0.05).

HSF genes
Changes in mean mRNA expression levels of HSF1, HSF2, and HSF4 in soleus muscle during the experimental period are shown in Figure 5. HSF1 gene was not detected in HSF1-null mice. In wild-type mice, there were no significant changes associated with both growth and overloading in mRNA expression levels of HSF1, HSF2, and HSF4. On the other hand, significant increases were induced in mRNA expressions of HSF2 (4th week) and HSF4 mRNA (2nd and 4th week) by overloading compared with the contralateral control level in HSF1-null mice, respectively (p,0.05). Significantly different HSF2 mRNA expression in control muscles between wild-type and HSF1-null mice was noted at the 4th week (p,0.05).
In both wild-type and HSF1-null mice, mRNA expression level of HSP25 increased following 2 weeks of overloading (p,0.05, Fig. 6). After 4 weeks of overloading, significant up-regulation of HSP25 mRNA was still noted in HSF1-null (p,0.05), but not in wild-type, mice. The levels of HSP25 mRNA expression in the overloaded muscles in wild-type mice were significantly greater than in HSF1null mice after 2 weeks of overloading (p,0.05). HSP47 mRNA expression was significantly up-regulated in HSF1null mice following 2 weeks of overloading (p,0.05), but not in wild-type mice. There were no significant changes in mRNA expression level of HSP47 in both types of mice after 4 weeks of overloading. Overloading-associated up-regulation was not inducted in mRNA expressions of HSC70, HSP72, and HSP90a in both wild-type and HSF1-null mice.
Representative expression patterns of HSP25, HSP47, HSC70, HSP72, HSP90a and GAPDH proteins are shown in Figure 7A. Protein expression levels of HSP25 (2nd week) and HSC70 (2nd and 4th week) of the contralateral control soleus muscle in HSF1null mice were significantly lower than those in wild-type mice (Fig. 7B, p,0.05). Significant increases in protein expression levels of HSP25, HSP47, and HSC70 were observed in both wild-type and HSF1-null mice following 2 weeks of overloading (p,0.05). The mean expression levels of HSP 25 and HSC70, but not of HSP47, in wild-type mice were significantly higher than those in HSF1-null mice following 2 weeks of overloading (p,0.05).
Increased expression levels of HSP25 protein were also observed in both wild-type and HSF1-null mice following 4 weeks of overloading (Fig. 7B, p,0.05). The mean expression level of HSC70 protein in wild-type mice was significantly higher than that in HSF1-null mice (p,0.05). There was no significant difference in the protein expression levels of HSP47 following 4 weeks of overloading. Overloading-related changes in protein expression levels of HSP72 and HSP90a were not observed in both HSF1-null and wild-type mice, either. Figure 8 shows the representative responses of phosphorylated Akt (p-Akt), total Akt (t-Akt), and the mean level of p-Akt relative to t-Akt (p-Akt/t-Akt). Relative expression levels of p-Akt to t-Akt in control muscle were similar between wild-type and HSF1-null  mice. Significant increases in the relative expression of p-Akt were observed in both wild-type and HSF1-null mice following 2, but not 4, weeks of overloading (p,0.05).

Pro-inflammatory cytokines
Changes in mean mRNA expression levels of pro-inflammatory cytokines, IL-6, ATF3, IL-1b, and TNF, in soleus muscle during the experimental period are shown in Figure 9. The levels of ATF3, but not of IL-6, IL-1b, and TNF, in control muscle of wildtype mice at the second week of experimental period was greater than those of HSF1-null mice (p,0.05). Significant up-regulations of IL-6 and ATF3 mRNAs were observed in both wild-type and HSF1-null mice following 2 weeks of overloading (p,0.05). The expression level of AFT3 mRNA in overloaded muscle of wild-type mice was greater than of HSF1-null mice (p,0.05). Significant up-regulation of IL-6 mRNA in HSF1-null mice, but not in wild-type, was still observed after 4 weeks of overloading (p,0.05). On the other hand, the overloading-related upregulation of ATF3 mRNA expression was maintained in wildtype mice (p,0.05), but not in HSF1-null mice, after 4 weeks of overloading.
Expression levels of IL-1b and TNF mRNAs were also upregulated in HSF1-null, not in wild-type, mice following 2 weeks of overloading (p,0.05, Fig. 9). The expression levels of IL-1b and TNF in the overloaded soleus muscle of HSF1-null mice were greater than in wild-type mice (p,0.05). Significant up-regulation of IL-1b mRNA expression was still observed in HSF1-null mice  following 4 weeks of overloading, but not in wild-type mice (p,0.05).

Discussion
Present study showed that absence of HSF1 partially inhibited the overloading-associated increments of muscle wet weight, protein content, and fiber CSA of mouse soleus muscle. This is the first report showing the inhibitory effects of HSG1-deficiency on skeletal muscle hypertrophy. Overloading-induced increase of the population of Pax7-positive muscle satellite cells, which may play a key role in skeletal muscle hypertrophy, was also attenuated by HSF1-defiency. On the other hand, HSF1 did not affect overloading-related phosphorylation of Akt. HSF1-deficiency decreased HSP expressions at mRNA and protein levels.
Overloading-related up-regulations of not only HSF2 and HSF4 but also HSPs were observed in HSF1-deficient mice. Significant up-regulations of IL-1b and TNF mRNAs were observed in HSF1-null, not wild-type, mice following 2 weeks of overloading. Overloading-related increases of IL-6 and AFT3 mRNA expressions seen after 2 weeks of overloading tended to decrease after 4 weeks in both types of mice. In HSF1-null mice, however, the significant overloading-related increase in the expression of IL-6, not ATF3, mRNA was noted even at 4th week. Inhibition of muscle hypertrophy might be attributed to the greater and prolonged enhancement of IL-6 expression.

Effects of HSF1-deficiency on overloading-associated skeletal muscle hypertrophy
In the present study, HSF1-deficiency did not affect the population level of Pax7-positive satellite cells in the control muscle. This result is consistent with our previous study [23]. But the overloading-associated increases of Pax7-positive satellite cells were inhibited in HSF1-null mice with restrained muscle hypertrophy. Although molecular mechanism(s) for HSF1-mediated regulation of satellite cells remains unclear, HSF1 may play a part in the regulation of proliferative potential of Pax-7-associated muscle satellite cells in overloading-induced skeletal muscle hypertrophy.
It is still unclear whether the level of Pax7-positive satellite cells affects overloading-associated skeletal muscle hypertrophy or not. A conditional knock-out technique revealed that satellite cell depletion did not inhibit the regrowth of skeletal muscle from unloading-induced atrophy [40]. This report also suggested the regrowth of atrophied soleus did not require the addition of new nuclei, which source is considered to be satellite cells. It was reported that muscle satellite cells were not essential for mechanical loading-associated skeletal muscle [41]. However, it was also suggested that the increased myonuclei in hypertrophied muscle fibers were attributed to muscle satellite cells, since the increase of myonuclei was observed in overloading-associated muscle hypertrophy under physiological condition [41]. On the contrary, another report indicated that the rate of regrowth in mouse soleus muscle was proportional to the level of Pax7-positive satellite cells [2].
In the present study, lower regrowing rate of overloaded skeletal muscle in HSF1-null mice was observed, compared with wild-type mice. On the other hand, a significant increase in the population of Pax7-positive satellite cells in wild-type mice was observed 2 weeks of overloading, but not in HSF-null mice. Therefore, greater regrowing rate in wild-type mice might be attributed to higher level of Pax7-positive satellite cells. However, the Pax7positive satellite cells did not increase in HSF-null mice, even though muscle hypertrophy was observed following 4, but not 2, weeks of overloading. Physiological significance of the overloading-associated increase of Pax7-positive satellite cells is still unclear.
Mechanical overloading, as well as heat stress, has a stimulating effect on not only proliferative potential of skeletal muscle [2,33,35,42] but also up-regulations of HSPs [13,15,43]. However, it is still unclear whether overloading-associated skeletal muscle hypertrophy is attributed to the chaperonic action of HSPs or stress-associated stimulation of protein synthesis. Although HSF1deficiency modified the expressions of HSPs at mRNA and protein levels in the present study, the changes in HSP mRNAs were inconsistent with those in HSP proteins. These results were consistent with those in the previous study [16]. It has been also suggested that post-translational regulation plays a role in the regulation of HSP protein levels in skeletal muscle [44246].
Lower expressions of HSP25 and HSC70 proteins in HSF1deficient mice were noted in the current study. Overloadingassociated up-regulations of HSP25 and HSC70 proteins were also attenuated by HSF1-deficiency. It has been reported that overexpression of HSP25 or HSP70 inhibits the immobilizationinduced muscle atrophy via attenuation of increase in muscle-  specific E3 ubiquitin ligases during disuse [10,11]. In addition, drug-induced up-regulation of HSP72 stimulated differentiation and protein synthesis in C2C12 myoblasts [47]. Impaired regeneration of skeletal muscle related to deficiency of HSF1 [23] or inducible HSP70 [48] was also reported. Although there is no report regarding the effects of lower expression levels of HSP25 and HSC70 on skeletal muscle hypertrophy, lower expressions of these HSP proteins may be one of the causes for the inhibition of skeletal muscle hypertrophy.
HSF1-deficiency did not affect the expression level of HSP47 protein in normal soleus muscle. Up-regulation of HSP47 protein in association with muscle hypertrophy was noted regardless of the presence of HSF1 gene in the resent study. Physiological role of collagen-specific HSP47 in skeletal muscle hypertrophy is not unclear. Up-regulations of HSP47 proteins have been reported during regeneration of injured mouse soleus muscle [23] and in soleus muscle of rats exposed to hypergravitational environment [18]. HSP47, as well as HSP72 and HSP90a, may not have any role(s) in overloading-associated skeletal muscle hypertrophy, since there were no changes in the expression levels of HSP72 and HSP90a proteins.

Possible effects of HSF1 on pro-inflammatory cytokines during skeletal muscle hypertrophy
In the present study, up-regulation of pro-inflammatory cytokines was observed following 2-week of overloading on soleus muscle. Previous studies showed that pro-inflammatory cytokines were up-regulated by increased workload, such as muscle contraction and exercise [25,49253]. The present study demonstrated that expression levels of IL-6 and ATF3 mRNAs were upregulated regardless of the presence of HSF1 gene. However, prolonged up-regulation of IL-6 mRNA by overloading was observed HSF1-deficient mice. On the contrary, overloadingassociated up-regulation of ATF3 mRNA was enhanced by the presence of HSF1. Overloading-associated up-regulation of IL-6 may be depressed by HSF1-associated up-regulation of ATF3 [24] in skeletal muscle. Although it has been suggested that IL-6 plays a significant role in muscle satellite cell-mediated skeletal muscle hypertrophy [54,55], the role(s) of the prolonged up-regulation of IL-6 and ATF3 in overloading-associated skeletal muscle hypertrophy remains unclear.
In the present study, HSF1-deficiency also enhanced the upregulation of TNF and IL-1b mRNAs in response to overloading. As IL-1b and TNF function as the inhibitors of myogenic differentiation [27229], enhanced up-regulations of these proinflammatory cytokines may be one of the causes for the inhibition of overloading-associated skeletal muscle hypertrophy in HSF1null mice. Although TNFa signaling impairs insulin/insulin-like growth factor signaling by Akt [30,31], a significant effect of presence or absence of HSF1 gene in p-Akt level was not observed. Further studies are essential to elucidate the regulatory roles of pro-inflammatory cytokines in overloading-associated skeletal muscle hypertrophy.
Effects of HSF1-deficiency on the expression level of HSPs in response to overloading Up-regulation of HSPs in mammalian skeletal muscle is mediated by HSF1 [21,22]. However, up-regulations of HSPs were observed in both wild-type and HSF1-deficient mouse skeletal muscles in the present study. It was confirmed that HSF2 and HSF4 in HSF1-null mouse soleus muscle were up-regulated by 2 or 4 weeks of functional overloading, but not in wild-type mice. The up-regulation of these HSFs was also observed in response to reloading of atrophied soleus muscle in HSF1-null mice [16]. HSPs could be up-regulated by mechanical loading via HSF2 and/or HSF4, even if HSF1 gene is absent.
HSF2 content in cultured C2C12 mouse myoblasts (or myotubes) is transiently up-regulated by initiation of myogenic differentiation, suggesting that HSF2 may play a role in skeletal muscle development [56]. Both significant increase in muscle weight and HSF2 mRNA expression of overloaded soleus muscle in HSF1-null mice were observed following 4 weeks of overloading. Therefore, it is speculated that up-regulation of HSF2 might be a phenomenon caused as the compensation for HSF1deficinecy, and cause the HSF2-dependent muscle hypertrophy in HSF1-null mice following 4 weeks of overloading.
Although expression of HSF4 mRNA is confirmed in skeletal muscle [57], HSF4 protein is detected in C2C12 mouse myoblasts, but not in B6D2F1 (BDF1) hybrid mouse skeletal muscle [58]. Even though human HSF4 functions to repress the expression of endogenous HSP27, HSP70, and HSP90 genes [57], there is no report concerning a physiological role of HSF4 in skeletal muscle. Up-regulations of HSF4 might be also a compensation for HSF1deficinecy. Further studies are needed to elucidate a physiological role of HSF2 and HSF4 in skeletal muscle hypertrophy.
In conclusion, HSF1-deffiency inhibited overloading-associated skeletal muscle hypertrophy in mice. Loading-related-increase in Pax7-positive satellite cells was also attenuated by absence of HSF1. Inhibition of muscle hypertrophy in HSF1-null mice may be related to the enhanced up-regulation of pro-inflammatory cytokines, such as IL-6, IL-1b, and TNF, that act as inhibitors on myogenic differentiation. In addition, up-regulations of HSPs were inhibited in mice without HSF1 gene. HSF1 and/or HSF1mediated stress response may play a key role in the overloadingassociated skeletal muscle hypertrophy.