3.1 KDM3A expression in CFs.
Firstly, we identified the phenotype and purity of CFs after 2-3 generations. Previous studies demonstrated that vimentin was surface-specific antigen of cardiac fibroblasts[14], and α-SMA antibodies was surface marker of myofibroblasts[15]. As shown in Figure 1a, we successfully isolated highly purified primary rat CFs by immunofluorescence analysis. Ninety-five percent of cells were stained with vimentin (red), almost no cells were stained with α-SMA (green), indicating that the isolated CFs could be utilized for subsequent experiments.
Next, we constructed relevant adenovirus to administered KDM3A expression. CFs were infected with KDM3A(AdKDM3A) or siRNA-KDM3A(AdshKDM3A). The results showed CFs transfected with AdKDM3A or AdshKDM3A (Figure 1b) emitted strong green fluorescence (GFP), indicating that target virus vectors were successfully transfected into the CFs. Then, we further measured the protein expression of KDM3A by Western blot analysis, and the results indicated that KDM3A expression was significantly enhanced after transfection with AdKDM3A, while AdshKDM3A transfection blocked KDM3A expression (Figure 1c-d). These data indicated that the target adenovirus we constructed effectively regulated the protein expression of KDM3A in CFs.
3.2 TGFβ1 induces cardiac fibroblast differentiation and significantly impacts KDM3A expression.
As we all know, TGFβ/Smad signaling as a classic profibrotic pathway that increase fibroblast abundance and cardiac fibroblast differentiation is a key event in cardiac remodeling[16, 17]. Moreover, TGFβ1 is the most common subtype of TGFβ. Thus, firstly, we verified the effects of TGFβ1 on cardiac fibroblast differentiation, and expression levels of vimentin, DDR2, α-SMA and CTGF were used as standard indexes of cardiac fibroblast differentiation[18]. Figure 2a-b showed that CTGF and α-SMA were significantly upregulated, while Vimentin and DDR2 were significantly downregulated response to TGFβ1 treatment for 24 h. As expected, we demonstrated that TGFβ1 could induce cardiac fibroblast differentiation. Importantly, consistent with the previous researches, KDM3A might participated in cardiac fibrosis[19]. In present study, we found that compared to control group, KDM3A level was significantly elevated (Figure 2c-d). Taken together, these results suggest that TGF-β1 promotes cardiac fibroblast differentiation and significantly enriches KDM3A expression. Based on the above observations, we speculated that KDM3A might be a key regulation factor during the course of cardiac fibroblast differentiation. To manifest this hypothesis, KDM3A loss- and gain- of- function assays were preformed in CFs.
3.3 KDM3A disruption significantly influences CFs proliferation and migration.
Based on above observations, we further explored whether KDM3A can intervene in specific biological functions of fibroblasts. First, we designed CCK-8 and migration experiments to evaluate fibroblast proliferation and migration function, respectively. The results showed that loss of KDM3A markedly suppressed CFs proliferation and migration under the stimulation of TGF-β1 for 24 h. (Figure 3a, c, d); however, when KDM3A was upregulated, the opposite tendency was observed, CFs proliferation and migration were significantly increased (3b, c, d). Collectively, suppression of KDM3A in CFs could abrogate the proliferation and migration, but overexpression of KDM3A had the opposite consequences.
3.4 KDM3A is a positive regulator of cardiac fibroblast differentiation, but KDM3A inhibition can reverse myofibroblast conversion.
To determine whether KDM3A is involved in cardiac fibroblast differentiation, we analyzed the changes in α-SMA, collagen I, and CTGF expression in response to changes in KDM3A expression. CFs were transfected with siRNA-KDM3A to knock down its expression, and transfected with AdKDM3A to active its expression. The results showed that CFs conversion was decreased by KDM3A downregulation that the expression of collagen I, CTGF and α-SMA in fibroblast was significantly lower than that in the control group under the stimulation of TGF-β1 for 24 h (Figure 4a). Consistent with the previous studies, after KDM3A upregulation, the conversion of CFs was obviously aggravated (Figure 4b). These data indicate that KDM3A inhibition could protect CFs against the conversion, but overexpression of KDM3A could further exacerbate CFs differentiation.
3.5 KDM3A regulates the biological function of CFs through the Smad3-dependent manner.
It is obviously noted that cardiac fibroblast differentiation is pivotal for the pathogenesis of cardiac fibrosis and hypertrophy[11, 20]. However, the underlying molecular mechanisms of cardiac fibroblast differentiation are still confused. Since CFs were featured with an enhanced expression of KDM3A induced by TGF-β1 for 24 h (Figure 2c), we further illustrated KDM3A participate in regulating functionalities of CFs (Figure 3). Additionally, we found that high expression of KDM3A could also simultaneously increase Smad3 phosphorylation, and Smad3 level had no significant change (Figure 4d). Nevertheless, after knocked down KDM3A by transfected siRNA-KDM3A, Smad3 phosphorylation was suppressed evidently by treated with TGF-β1(Figure 4c). These findings prompted us to assume that KDM3A may be induced by TGF-β1 signaling, and then regulating functionalities of CFs by Smad3 signaling.
To confirm this hypothesis, CFs were treated with SIS3-HCL (a specific inhibitor of p-Smad3), followed by TGF-β1 induction. It was remarkably noted that p-Smad3 deficiency by SIS3-HCL could both significantly abolish KDM3A positive effect, which was accompanied with weakened cardiac fibroblast differentiation related proteins (collagen I, CTGF, α-SMA) induced by TGF-β1(Figure 4b, d), indicating that Smad3 were indispensable factors for KDM3A alter cardiac fibroblast differentiation in response to TGF-β1. Moreover, consistent with the results of cardiac fibroblast differentiation, we found that p-Smad3 inhibition by SIS3-HCL was indeed impair CFs proliferation and migration ability (Figure 3 b,c,d). Altogether, these data corroborated that overexpression of KDM3A aggravated cardiac fibroblast differentiation induced by TGF-β1 was a Smad3-dependent manner.