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Transmembrane proteoglycans syndecan-2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early-differentiated myoblasts

Physiological Reports (2015) 3(9)
DOI: 10.14814/phy2.12553
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Abstract

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed an unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) triggered its expression exclusively at the early differentiation phase. In order to advance this concept, the present study described that transmembrane heparan/chondroitin sulfate proteoglycans syndecan-2, 4 may be the plausible receptor candidates for HGF and FGF2 to signal Sema3A expression. Results showed that mRNA expression of syndecan-2, 4 was abundant (two magnitudes higher than syndecan-1, 3) in early-differentiated myoblasts and their in vitro knockdown diminished the HGF/FGF2-induced expression of Sema3A down to a baseline level. Pretreatment with heparitinase and chondroitinase ABC decreased the HGF and FGF2 responses, respectively, in non-knockdown cultures, supporting a possible model that HGF and FGF2 may bind to heparan and chondroitin sulfate chains of syndecan-2, 4 to signal Sema3A expression. The findings, therefore, extend our understanding that HGF/FGF2-syndecan-2, 4 association may stimulate a burst of Sema3A secretion by myoblasts recruited to the site of muscle injury; this would ensure a coordinated delay in the attachment of motoneuron terminals onto fibers early in muscle regeneration, and thus synchronize the recovery of muscle fiber integrity and the early resolution of inflammation after injury with reinnervation toward functional recovery.

Figures

  • Figure 1. Null effect of FGFR1 immunoneutralization on FGF2induced Sema3A upregulation. Satellite cells (over 95% desminpositive at 30 h postplating) were isolated from back and upper hind-limb muscles of 9-month-old male Sprague Dawley rats according to Allen et al. (1997) and maintained in DMEM-10% HS for 48 h. Myoblasts were then pretreated with 0–3.0 lg/mL mouse monoclonal anti-FGFR1 neutralizing antibody (LS-C23657; Lifespan BioSciences, Washington, DC) (Kang et al. 2012) in DMEM-10% HS for 90 min prior to adding recombinant FGF2 (2.5 ng/mL) to the media. Cells were maintained for the next 24 h, and evaluated for Sema3A expression (closed circle) by real-time RT-qPCR run under the TaqMan probe detection format, standardized to the expression of HPRT. Inset, PCNA expression in the same cultures; note that the cell proliferation activity was reduced by the immunoneutralization even in the presence of FGF2 (solid bar), and hence this treatment served as an important positive control. Open circle and bar labeled “CNT” represent the negative control culture without the neutralizing antibody or FGF2 addition. Data points and bars depict the means SEs for three cultures per treatment and significant differences from the positive control culture that received 2.5 ng/ mL FGF2 (without anti-FGFR1 antibody) at P < 0.05 and P < 0.01 are indicated by (*) and (**), respectively.
  • Table 1. Stealth siRNA strands for mouse SDC1–4.
  • Figure 2. Knockdown potential of SDC isoform-specific siRNAs in myoblast cultures. Myoblast cultures were transfected with each siRNA at 100 nmol/L, as follows: control siRNA (black bars) and SDC1–4 siRNA (colored bars; Stealth_384, 715, 789, and 251, respectively) in DMEM-5%HS without HGF/FGF2, followed by evaluation of the level of expression of SDC1–4mRNA at 24 h by real-time qPCR standardized to HPRT. Data bars depict the means SEs for three cultures per treatment; significant differences from control siRNA culture means at P < 0.01 are indicated by (**).
  • Table 2. qPCR primer sets for Sema3A, SDC1–4, and references
  • Figure 3. Expression profiles of SDC members in skeletal muscle, differentiated myoblasts, and referenced organs and tissues. Tibialis anterior (TA) and gastrocnemius muscles (Gas) of lower hind-limb, and cardiac muscle (not whole-heart tissue), lung, liver, and kidney were all collected from 10-week-old male C57BL/6J mice. Satellite cells (SCs) were isolated from muscle groups of upper hind-limb and back of the same mice and maintained in DMEM-10% HS until 72-h postplating – the early differentiation time point is as described previously (Tatsumi et al. 2009; Do et al. 2011). Mouse satellite cell-derived myoblasts (myoblasts) were also cultured for 72 h in DMEM-5% HS differentiation medium. Relative levels of mRNA expression of SDC members (SDC1–4) were determined by real-time RT-qPCR combined with the 2 DDC(t) analysis referenced to b-actin expression. Data bars depict the means SEs for three mice on a log scale.
  • Figure 4. SDC2, 4 may mediate FGF2-induced Sema3A upregulation in myoblasts. Myoblast cultures were transfected with each siRNA at 100 nmol/L, as follows: control siRNA (black bars) and SDC1–4 siRNA (colored bars; Stealth_384, 715, 789, and 251, respectively) in DMEM-5% HS with 2.5 ng/mL FGF2 (see an upper scheme shown in panel A for the culture design), followed by evaluation for expression levels of SDC1–4 and Sema3A at 72 h by RT-qPCR (panel A) and western blotting (panel B; a representative cell lysate sample from three cultures per treatment group normalized to b-actin expression levels). Note that there were no significant differences (NS) in Sema3A expression levels between the negative control (open bar, without siRNA and FGF2 addition) and the cultures transfected with SDC2, 4 siRNAs, as well as between the positive control (gray bar, treated with 2.5 ng/mL FGF2 without control siRNA) and the experimental control cultures (black bars and CNT, treated with both 100 nmol/L control siRNA and 2.5 ng/mL FGF2). Data bars depict the means SEs for three cultures per treatment and significant differences from the experimental control culture mean at P < 0.05 and P < 0.01 are indicated by (*) and (**), respectively. STD, molecular weight standards.
  • Figure 5. SDC2, 4 may mediate HGF-induced Sema3A upregulation in myoblasts. Myoblast cultures were transfected with each siRNA at 100 nmol/L, as follows: control siRNA (black bars) and SDC1–4 siRNA (colored bars; Stealth_384, 715, 789, and 251, respectively) in DMEM-5% HS with 25 ng/mL HGF (the same culture design as Fig. 4), followed by evaluation for expression levels of SDC1–4 and Sema3A at 72 h by real-time RT-qPCR (panel A) and western blotting (panel B; a representative sample from three cultures per each treatment group normalized to b-actin expression levels). Note that there were no significant differences (NS) in the level of Sema3A expression between the negative control (open bar, without siRNA and HGF addition) and the cultures transfected with SDC2, 4 siRNAs, or between the positive control (gray bar, received 25 ng/mL HGF without control siRNA) and the experimental control cultures (black bars and CNT, received both 100 nmol/L control siRNA and 25 ng/mL HGF). Data bars depict the means SEs for three cultures per treatment and significant differences from the experimental control culture mean at P < 0.05 and P < 0.01 are indicated by (*) and (**), respectively. STD, molecular weight standards.
  • Figure 6. Positive effects of glycosaminoglycan chain hydrolysis on HGF/FGF2-induced Sema3A upregulation. Primary cultures of rat satellite cells were maintained in DMEM-10% HS for 48 h and then pretreated with one of the GAG-chain hydrolyzing enzymes, heparitinase (0.5–40 mU/mL) from Flavobacterium heparinum (100703; Seikagaku, Tokyo, Japan) (A, B) or chondroitinase ABC (1–100 mU/mL) from Proteus vulgaris (100332; Seikagaku) (C, D) in serum-free DMEM, for 90 min before a 24-h period of supplementation with 25 ng/mL HGF (A, C) and 2.5 ng/ mL FGF2 (B, D). Cell lysates were collected and analyzed for expression of Sema3A (closed symbols) and proliferating cell nuclear antigen (PCNA; panels A and D, insets) by real-time RT-qPCR standardized with HPRT. Open symbols and bars labeled “CNT” (far left) represent negative control cultures without any treatment. Closed symbols labeled “HGF” and “FGF2” represent positive control cultures without enzyme treatment and with 25 ng/mL HGF and 2.5 ng/mL FGF2, respectively. Data points and bars depict the means SEs for three cultures per treatment and significant differences from each positive control culture mean at P < 0.05 and P < 0.01 are indicated by (*) and (**), respectively. Note that the heparitinase/chondroitinase ABC treatments do not significantly disturb the cell viability (P > 0.05) as monitored by the mRNA expression level of PCNA, in the enzyme–dose ranges examined (panels A and D, insets).

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Do, M. K. Q., Shimizu, N., Suzuki, T., Ohtsubo, H., Mizunoya, W., Nakamura, M., … Tatsumi, R. (2015). Transmembrane proteoglycans syndecan-2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early-differentiated myoblasts. Physiological Reports, 3(9). https://doi.org/10.14814/phy2.12553

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