Abstract
Well-regulated differentiation of fibroblasts into myofibroblasts (MF) is critical for skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), an established marker for MF differentiation, is driven by TGFβ receptor (TGFβR)-mediated signaling. Hyaluronan (HA) and its receptor CD44 may also participate in this process. To further understand this process, primary mouse skin fibroblasts were isolated and treated in vitro with recombinant TGF-β1 (rTGF-β1) to induce α-SMA expression. CD44 expression was also increased. Paradoxically, CD44 knockdown by RNA interference (RNAi) led to increased α-SMA expression and α-SMA-containing stress fibers. Removal of extracellular HA or inhibition of HA synthesis had no effect on α-SMA levels, suggesting a dispensable role for HA. Exploration of mechanisms linking CD44 knockdown to α-SMA induction, using RNAi and chemical inhibitors, revealed a requirement for noncanonical TGFβR signaling through p38MAPK. Decreased monomeric G-actin but increased filamentous F-actin following CD44 RNAi suggested a possible role for myocardin-related transcription factor (MRTF), a known regulator of α-SMA transcription and itself regulated by G-actin binding. CD44 RNAi promoted nuclear accumulation of MRTF and the binding to its transcriptional cofactor SRF. MRTF knockdown abrogated the increased α-SMA expression caused by CD44 RNAi, suggesting that MRTF is required for CD44-mediated regulation of α-SMA. Finally, chemical inhibition of p38MAPK reversed nuclear MRTF accumulation after rTGF-β1 addition or CD44 RNAi, revealing a central involvement of p38MAPK in both cases. We concluded that CD44 regulates α-SMA gene expression through cooperation between two intersecting signaling pathways, one mediated by G-actin/MRTF and the other via TGFβR/p38MAPK.
Highlights
Well-regulated differentiation of fibroblasts into myofibroblasts (MF) is critical for skin wound healing
Blockade of EGFR or ERK1/2 did nothing to blunt the effect of CD44 knockdown, and ERK1/2 phosphorylation was unaffected by CD44 RNA interference (RNAi), suggesting no involvement of EGFR signaling
Regarding TGF receptor (TGFR) signaling, we found that CD44 knockdown did not alter the phosphorylation of Smad2/3, and had only mild effects on phosphorylation of p38MAPK, suggesting that CD44 RNAi does not directly modulate TGFR activation
Summary
CD44s is the predominant CD44 isoform in primary murine dermal fibroblasts and is inhibitory to ␣-SMA gene expression. Consistent with the earlier findings using immunofluorescence (Fig. 5, A and B), the changes in Fig. 5C were most pronounced when CD44 RNAi and rTGF-1 were administered together It has been demonstrated by various groups that nuclear localization of MRTF and its binding to SRF play a crucial role in regulating the transcription of contractile genes including ␣-SMA [11, 39, 40]. Knockdown of MRTF by RNAi (knockdown efficacy was verified by Western blot analysis as shown in Fig. 7B) significantly suppressed the expression ␣-SMA in the absence or presence of rTGF-1 and effectively abrogated CD44 RNAi’s ability to induce ␣-SMA gene expression (Fig. 7, A and C). RNAi and TGF-1 upon subcellular localization of MRTF are dependent on p38MAPK activity
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