TRPC6 promotes fibroblast to myofibroblast transdifferentiation
To identify regulators of myofibroblast transdifferentiation we utilized the mammalian genome collection of expressed cDNAs (expression plasmids) to perform a cell-based gain-of-function screen in SV40 transformed mouse embryonic fibroblasts (MEFs). We utilized an αSMA-luciferase promoter plasmid as a transcriptional-program surrogate for myofibroblast activation. Less than 1% of 18,400 full-length cDNAs screened elicited a signal greater or equal to that obtained with recombinant TGFβ treatment. Two of the positive cDNAs identified were the known fibroblast activator interleukin-6 (IL6) and TRPC6 (). We confirmed the fibroblast-activating effect with a second luciferase reporter plasmid containing the periostin (Postn) promoter, which is also induced in transformed fibroblasts. TRPC6 overexpression induced both reporters similar to TGFβ treatment ().
Figure 1 TRPC6 overexpression promotes fibroblast to myofibroblast conversion. (A) αSMA-luciferase and Postn-luciferase promoter activity from cultured neonatal rat cardiac fibroblasts transfected with expression vectors encoding TRPC6 or IL6, or treated (more ...)
To investigate if TRPC6 can directly induce fibroblast to myofibroblast transdifferentiation we infected MEFs, primary rat cardiac fibroblasts and primary human dermal fibroblasts with a recombinant adenovirus (Ad) expressing TRPC6 or Adβgal (control) and then assayed for αSMA stress fiber formation and collagen I by confocal microscopy 48 hrs later (). AdTRPC6 infection induced αSMA stress fiber positivity and conversion of approximately 50% of all three types of fibroblasts, in a manner similar to TGFβ or AngII treatment, while less than 5% of control fibroblasts expressed αSMA (). AdTRPC6 infection also enhanced expression of the myofibroblast specific ED-A isoform of fibronectin by western blotting (Fn-EDA, ). As myofibroblasts contract a collagen gel matrix (Rice and Leinwand, 2003
; Tomasek et al., 2002
), AdTRPC6 infected fibroblasts or TGFβ treated showed contraction over 36 hrs while Adβgal infected controls did not (). However, the contraction of collagen gel matrices induced by TRPC6 overexpression was not due to altered cardiac fibroblast proliferation ().
TRPC channels form homo- or heterotetramers although overexpression of any one subunit alone can enhance Ca2+
currents (Eder and Molkentin, 2011
). To determine if myofibroblast transformation was specific to TRPC6 we also evaluated the ability of AdTRPC3 and AdTRPC4 infection to induce αSMA expression and contract collagen matrices in primary cardiac fibroblasts (Figure S1A, S1B, and S1C
). TRPC4 showed no effect while TRPC3 overexpression weakly converted some fibroblasts to myofibroblasts, although not nearly to the same magnitude as TRPC6.
Loss of TRPC6 prevents TGFβ-mediated myofibroblast transformation
To determine if TRPC6 is required for myofibroblast conversion, dermal fibroblasts isolated from Trpc6
wildtype (Wt) and null littermates (Dietrich et al., 2005
) were examined for TGFβ induction of αSMA stress fiber formation and contractile function. Remarkably, Trpc6−/−
primary dermal fibroblasts showed no induction of αSMA positive stress fibers with TGFβ treatment in contrast to robust induction in similarly prepared Wt fibroblasts (). Moreover, TGFβ induced profound contraction of collagen gels cultured with Wt fibroblasts, while Trpc6−/−
fibroblasts were refractory to TGFβ-mediated contraction (). Interestingly, the ability to repopulate a scratched confluent monolayer in culture was either not impaired in Trpc6−/−
fibroblasts or was more proficient compared with Wt fibroblasts, depending on culturing conditions ().
Figure 2 Loss of TRPC6 prevents TGFβ-mediated myofibroblast conversion. (A) Immunofluorescent staining of αSMA (green) positive stress fibers and TOPRO-3 iodide nucleic acid stain (blue) in primary Trpc6+/+ (Wt) and Trpc6−/− dermal (more ...)
TRPC6 is necessary for dermal and cardiac wound healing
Here a dermal wound-healing model was employed in which 2 dorsal 6 mm punch biopsies were taken from Trpc6 Wt and null littermates. Wounds were first generated in Wt mice and the healing border areas were dissected at day 4 for RT-PCR analysis of TRPC6 mRNA. TRPC6 mRNA was dramatically induced in the wounds from 3 separate mice compared with lower levels in 2 uninjured control mice (). Consistent with these results, Trpc6−/− mice had significantly impaired wound closure rates over time such that by day 5 when Trpc6 Wt mice had highly retracted wounds, Trpc6−/− mice attained only 33.1% retraction (). At 3 days post biopsy the border zones of the wounds were stained for αSMA (myofibroblasts) and isolectin (endothelial cells) (). Trpc6−/− histological sections had significantly less myofibroblasts per area than Wt counterparts suggesting that the poor dermal wound healing in Trpc6−/− mice could be due to impaired myofibroblast formation ().
Figure 3 TRPC6 is necessary for dermal and cardiac wound healing. (A) RT-PCR for TRPC6 mRNA from skin wounds of 3 separate mice or uninjured skin of 2 control mice. GAPDH is used as a control. (B) Photographs of 2 full excision 6 mm dorsal coat (skin) punch biopsies (more ...)
We also examined a cardiac injury model involving myocardial infarction (MI), as myofibroblast transformation is critical to maintain ventricular wall structural integrity and to reduce dilation. Compared with Wt mice, Trpc6−/− mice had significantly higher rates of mortality due to ventricular wall rupture throughout 3–7 days post MI injury, a time during which scar formation and remodeling mostly occurs (). The Trpc6−/− mice that survived MI had significantly smaller scar sizes, greater reductions in cardiac function, and significantly greater ventricular wall dilation compared with Wt MI mice ().
Non-canonical TGFβ signaling induces TRPC6 gene expression to activate myofibroblasts
TGFβ stimulation induced mRNA levels of TRPC6 in primary cardiac fibroblasts but not in other TRPC family members, except for a slight increase in TRPC1 (). Immunofluorescent analysis of cardiac fibroblasts fixed 48 hrs after TGFβ treatment also showed enhanced TRPC6 protein levels, similar to AdTRPC6 infection (). Since TGFβ can mediate its effects through canonical (SMAD2/3) or non-canonical (MAPK) pathways we examined these downstream effects. The TGFβ-dependent induction of TRPC6 expression was not inhibited by infection with AdSMAD6 or AdSMAD7, known inhibitors of canonical signaling (). However, the p38 MAPK inhibitor SB731445 completely blocked TGFβ-dependent induction of TRPC6 expression suggesting a critical role for non-canonical signaling in TRPC6 induction.
Figure 4 Non-canonical TGFβ-mediated signaling induces TRPC6 gene expression to activate myofibroblasts. (A) Representative RT-PCR for TRPC family members in which PCR product was sampled every 5 cycles starting at cycle 20 from cardiac fibroblasts ± (more ...)
Signaling by p38 MAPK is elicited by many different inflammatory cytokines (not just TGFβ signaling), such as through AngII and its receptor, which also fully induced TRPC6 gene expression in a p38-dependent manner (). This was particularly interesting because p38 signaling might represent a common intracellular event in myofibroblast transformation between diverse inflammatory cytokines and their different receptors. Indeed, TGFβ mediated αSMA stress fiber positivity and myofibroblast transformation was inhibited by blockade of p38 signaling, but not by AdSMAD7 ().
Importantly, shRNA mediated knockdown of the ALK5 (TGFBRI) receptor that fully suppressed TGFβ-dependent myofibroblast transformation did not inhibit TRPC6-dependent transformation (). This result indicates that TRPC6 is downstream of the TGFβreceptor complex in mediating myofibroblast transformation, although it does not distinguish canonical versus non-canonical signaling. Finally, ALK5 deficient cardiac fibroblasts infected with AdMKK6, which purely drives p38 MAPK signaling, fully promoted αSMA positive stress fiber formation and myofibroblast transformation, similar to AngII treatment, indicating that non-canonical p38 mobilization is the critical aspect of TGFβ signaling in forming myofibroblasts. This result also explains how AngII, ET-1 and select other inflammatory cytokines can themselves drive myofibroblast transformation without eliciting TGFβ signaling or SMAD transcriptional responses (see discussion).
SRF directly induces TRPC6 gene expression downstream of TGFβ-p38 MAPK signaling
SRF and its transcriptional cofactors, myocardin and myocardin-related transcription factor (MRTF), are activated by TGFβ receptor signaling to facilitate αSMA gene expression and myofibroblast transformation (Chai et al., 2007
; Crider et al., 2011
; Sandbo et al., 2009
). Investigation of the mouse TRPC6 proximal promoter (−1187 to +1) identified several potential SRF binding sites, of which the most proximal promoter sequence at −123 to −114 was highly conserved (Benson et al., 2011
). Here we determined that SRF overexpression in fibroblasts, but not the related transcription factor myocyte enhancer factor 2a (MEF2a), upregulated TRPC6 mRNA expression, which was blocked with the p38 inhibitor SB731445 (). Transfection of a luciferase reporter plasmid containing a 1187 base pair TRPC6 proximal promoter showed induction by TGFβ, SRF, and synergistic activation with both together (). A TRPC6 promoter deletion construct (−215) containing the conserved proximal SRF binding site (CArG box) at −123 to −114, showed over 3-fold induction with TGFβ stimulation, yet an identical promoter construct with this site mutated was unresponsive to TGFβ (Figure S2A
). The induction of TRPC6 promoter activity by SRF was blocked with the p38 inhibitor (). Importantly, chromatin immunoprecipitation (ChIP) experiments spanning −1238 to −855 bp of the TRPC6 promoter showed SRF binding that was induced with TGFβ but reduced with an adenovirus expressing an shRNA against SRF, suggesting direct regulation of TRPC6 gene expression (). Indeed, ChIP with an anti-phospho-SRF antibody (activated SRF) showed even greater inducible occupancy of the TRPC6 promoter upon TGFβ stimulation (). Similar results were achieved by ChIP with another primer pair that amplified the most proximal CArG element near the start site of transcription (data not shown). Gel shift analysis of the proximal −123 CArG box showed optimal binding of SRF from cell extracts that was blocked with prior infection with a recombinant adenovirus expressing an shRNA against SRF (Figure S2B
). Collectively, these results suggest that SRF can bind and directly regulate the TPRC6 promoter in a p38 dependent manner.
Figure 5 SRF mediates TRPC6 gene expression and myofibroblast transdifferentiation. (A) Real time PCR analysis of TRPC6 mRNA in primary cardiac fibroblasts infected with the indicated adenoviruses, with or without p38 inhibitor (SB731445). *P<0.05 vs Adβgal; (more ...)
AdSRF infection of primary Wt dermal fibroblasts fully induced myofibroblast transdifferentiation, as shown by immunocytochemistry for αSMA (). However, Trpc6−/−
dermal fibroblasts were completely refractory to SRF-mediated induction of myofibroblast transdifferentiation, indicating that TRPC6 is a required downstream mediator of SRF-dependent myofibroblast transformation (). Trpc6−/−
primary dermal fibroblasts were also refractory to TGFβ and AngII-mediated myofibroblast transdifferentiation, cytokines that are known to induce SRF or MRTF activity (Martin-Garrido et al., 2011
; Small et al., 2010
Finally, to examine the necessity of SRF in mediating TGFβ or AngII-dependent myofibroblast transdifferentiation we used a recombinant adenoviral construct expressing an shRNA against SRF (Streb and Miano, 2005
). Knock-down of SRF (>80% by western blot, ()) prevented both TGFβ and AngII-dependent myofibroblast transdifferentiation, but importantly, it did not inhibit transdifferentiation mediated by AdTRPC6 as assessed by αSMA staining or gel contraction assays (). Ad-shSRF infection of primary fibroblasts also reduced baseline TRPC6 mRNA expression, as well as blocked TGFβ induction of expression (Figure S2C
). These results place SRF in a critical intermediary position in the transdifferentiation signaling cascade, where it receives input from TGFβ and other profibrotic cytokines through p38 MAPK signaling, which then directly mediates TRPC6 gene expression to mobilize a Ca2+
-dependent pathway for transdifferentiation (see below).
TGFβ-p38 signaling enhances Ca2+ through TRPC6 in activated myofibroblasts
Given TRPC6’s role in Ca2+
entry, cardiac fibroblasts were either treated with TGFβ or infected with AdTRPC6 for 48 hrs and then SOCE was measured. Representative traces and quantification of the intracellular Ca2+
signal showed significant increases with AdTRPC6 and with 48 hrs of TGFβ and AngII compared to no treatment ( and Figure S3A
). This increase in SOCE was not due to acute TGFβ signaling since 15 minutes of ligand treatment had no effect, suggesting it was likely mediating transcriptional changes.
Figure 6 TRPC6-mediated Ca2+ signaling mediates myofibroblast transdifferentiation. (A) Quantification of store-operated calcium entry (SOCE) for control (no treatment or Adβgal infection, N=24), TGFβ treated (N=25), and AdTRPC6 (N=54) infected (more ...)
Relative to Wt dermal fibroblasts that demonstrated a nearly 2-fold increase in SOCE with TGFβ treatment, Trpc6−/− fibroblasts had no change, indicating that TRPC6 is the primary mediator of the TGFβ-dependent enhancement of SOCE in activated fibroblasts (). Moreover, this TGFβ-dependent increase in SOCE in fibroblasts was significantly reduced with a p38 MAPK inhibitor, while unitary activation of p38 with AdMKK6 infection induced a small, albeit significant increase in SOCE (). These results support the overall signaling model whereby non-canonical TGFβ signaling through p38 enhances myofibroblast transformation through a Ca2+ signal that uniquely depends on Trpc6 gene expression. However, these results do not establish that SOCE is the mechanism behind TRPC6’s ability to convert fibroblasts (SOCE was simply a surrogate assay for TRPC function), as TRPC channels can also mediate Ca2+ influx independent of store depletion (see discussion).
TRPC6-dependent Ca2+ induces calcineurin-NFAT for myofibroblast transformation
TRPC channels mediate their biologic effects primarily through Ca2+
influx and Ca2+
-dependent signaling effectors, of which calcineurin-NFAT has been particularly implicated (Kuwahara et al., 2006
). Indeed, nearly 50% of cardiac fibroblasts infected with a recombinant adenovirus expressing a constitutively active calcineurin (ΔCnA) mutant showed αSMA positive stress fibers similar to TRPC6 overexpression (). Coinfection of TRPC6 with the calcineurin-NFAT inhibitor Cain fully blocked this conversion while Cain alone had no effect (). Moreover, inhibition of calcineurin with Cain or specific inhibition of only NFAT with VIVIT blocked myofibroblast formation and collagen gel contraction in cardiac fibroblasts stimulated with TGFβ or AdTRPC6 (Figure S3B
(calcineurin B1) null MEFs (lack all calcineurin activity) were also unable to contract a collagen gel after TGFβ or AdTRPC6 infection (Figure S3C
). Taken together, these results indicate that activation of calcineurin is sufficient to drive myofibroblast transdifferentiation and that calcineurin signaling is necessary for TRPC6- and TGFβ-dependent formation of myofibroblasts.
TRPC6 overexpression caused a 12-fold increase in NFAT activity () that could be blocked by Cain (data not shown)
. AdΔCnA was used as a positive control yielding a 50-fold induction of NFAT activity (). TGFβ also elicited a significant increase in NFAT activity, although it was only 2-fold above baseline (). A more sensitive assay involving NFAT translocation showed a remarkable association between NFAT and TRPC6/TGFβ signaling. Specifically, NFATc1-GFP normally resides in both the cytoplasm and nucleus of quiescent fibroblasts, while infection with AdΔCnA sends all the NFATc1-GFP to the nucleus (). Both TGFβ and TRPC6 specifically sent essentially all of the NFATc1-GFP to the nucleus only in those fibroblasts that had converted to myofibroblasts with αSMA positivity (). Placing this result within the hierarchy of the larger signaling pathway, we also observed that AdΔCnA-induced myofibroblast transformation was independent of p38 MAPK since the inhibitor did not reduce αSMA positivity (). Moreover, inhibition of SRF with an shRNA against SRF did not block the ability AdΔCnA to induce myofibroblast transdifferentiation (). Finally, TGFβ stimulation also increased expression of calcineurin Aβ mRNA levels, which enhances total calcineurin activity (Figure S3D and S3E
Wt or Trpc6−/− fibroblasts infected with AdΔCnA each showed robust gel contraction, similar to AdTRPC6 infection (). As a control, TGFβ was still unable to elicit contraction in collagen gels containing Trpc6−/− fibroblasts ( and ). Collectively, these results indicate that calcineurin-NFAT signaling is downstream of the TGFβ-p38 MAPK-SRF signaling event that mediates enhanced TRPC6 expression/activity. These results define the ability of calcineurin to drive myofibroblast transformation as the downstream mediator or TRPC6 induction.
Figure 7 The TRPC6-Calcineurin-NFAT signaling axis is necessary for dermal wound healing. (A) Photographs and (B) quantification of floating collagen gel matrix contraction rates seeded prior with Trpc6+/+ or Trpc6−/− dermal fibroblasts at 48 hrs (more ...)
The TRPC6-calcineurin-NFAT signaling axis is necessary for dermal wound healing
Since ΔCnA overcame the defect in myofibroblast collagen gel contraction with loss of Trpc6
, we hypothesized that activated calcineurin would restore defective wound healing in Trpc6−/−
mice. To examine this hypothesis AdΔCnA, AdTRPC6 or AdGFP (as an internal control) was topically applied to dermal punch biopsies in Wt and Trpc6−/−
mice (). AdΔCnA significantly improved the rate of wound closure in Trpc6−/−
mice at each designated time point, similar to or even slightly better than AdTRPC6, while AdGFP infection had no effect (). Consistent with this result, Ppp3c−/−
(calcineurin Aβ) mice had significantly impaired dermal wound closure rates at days 4–7, although by day 10 healing eventually caught up to Wt controls (). Similarly, Wt mice treated with tacrolimus (FK506), a calcineurin inhibitor, showed a mild but significant impairment in wound healing, as previously suggested in the literature (Gisquet et al., 2011
; Schaffer et al., 1998
) (data not shown). By comparison, Trpc6−/−
mice had a more profound wound closure defect that never resolved even up to 10 days post injury ().