LG283 inhibits TGF-β-induced expression of ECM in human dermal fibroblasts
Excessive production of ECM by skin fibroblasts or myofibroblasts contributes to skin fibrosis. Therefore, we examined the biological effects of LG283, a small compound that was detected as a candidate for antifibrotic drug by our high-throughput in vitro screening, on ECM synthesis by cultured human normal skin fibroblasts. As assessed by real time-RT-PCR, baseline mRNA expression of COL1A2 and FN-1 was significantly increased by subsequent treatment with rTGF-β1 (Fig. 1a). In contrast, one-hour pretreatment with 4.5 μM of LG283 significantly suppressed the TGF-β1-dependent induction of both mRNAs to steady-state levels. A similar trend was observed for the protein expression of COL1A2 and FN-1, as assessed by western blotting (Fig. 1b). These findings suggest that pretreatment with LG283 efficiently inhibits TGF-β-induced fibrogenic activity of skin fibroblasts.
LG283 inhibits the TGF-β-dependent increase of transcription factors responsible for the mesenchymal transition of human dermal fibroblasts
The differentiation of fibroblasts into myofibroblasts is critical for local ECM production and resultant fibrosis in the skin. Therefore, we investigated the expression of representative transcription factors responsible for the differentiation into myofibroblasts in cultured, human normal skin fibroblasts (Fig. 1c). The mRNA expression of zinc-finger transcriptional regulators SNAIL1 and SNAIL2 was found to increase following treatment with rTGF-β1. However, pretreatment with LG283 significantly inhibited the TGF-β-dependent induction of both mRNAs. On the other hand, rTGF-β1 and/or LG283 did not alter the expression levels of ZEB1 and ZEB2, other zinc-finger transcriptional regulators associated with the transition into myofibroblasts, suggesting LG283 specifically inhibits the TGF-β-dependent mesenchymal transition cascade.
LG283 abrogates TGF-β-dependent phosphorylation of Smad3 in human dermal fibroblasts
The TGF-β binding to its receptor induces the phosphorylation of Smad2/3 transcription factors upon canonical signaling. Phosphorylated Smad2/3 and cytoplasmic Smad4 intercommunicate to transfer the signal to the nucleus and result in the transcriptional gene regulation responsible for tissue fibrosis. We investigated the effects of LG283 on Smad phosphorylation in cultured human normal skin fibroblasts. Immunocytochemical analysis revealed that treatment with rTGF-β1 increased cytoplasmic and nuclear staining for p-Smad3 (Fig. 1d). However, Smad3 phosphorylation was inhibited by pretreatment with LG283.
LG283 blocks TGF-β-induced EMT in cultured A549 lung epithelial cells
We performed an EMT assay using the A549 human lung carcinoma epithelial cell line. When cultured on 2-D plates, A549 cells rapidly grew to a confluent epithelioid sheet-like appearance, which was not affected by the presence of LG283 (Fig. 2a, upper panels). Morphologically, the cells appeared round with loose clusters and sparse intercellular adhesions. Upon treatment with rTGF-β2 for 72 h, the cells changed to a fibroblastic spindle shape (Fig. 2a, left lower). However, simultaneous treatment with rTGF-β2 and LG283 somewhat negated the morphological change of A549 cells (Fig. 2a, right lower). On 3-D culture, A549 cells rapidly formed colonies of various sizes similarly in the presence and absence of LG283 (Fig. 2b, upper panels). Treatment with rTGF-β2 caused the cells to spread out to form colonies and decreased the amount of intercellular adherence and size of each colony (Fig. 2b, left lower), all of which were inhibited by simultaneous treatment with LG283 (Fig. 2b, right lower).
Next, we examined whether the LG283-dependent morphological stability is linked with epithelial and mesenchymal gene expression in 3-D cultured A549 cells. Treatment with rTGF-β2 markedly reduced the expression of E-cadherin mRNA, a representative epithelial marker, but inversely increased expression of mesenchymal markers such as FN-1, α-SMA, and CTGF at 48hr (Fig. 2c). The altered expression pattern of epithelial and mesenchymal markers was significantly repressed by simultaneous treatment with LG283 (Fig. 2c). Treatment with TGF-β2 increased the expression of SNAIL1 and SNAIL2 mRNA, but not that of ZEB1 and ZEB2 (Fig. 2c). The increased mRNA expression of SNAIL1 and 2 was suppressed by simultaneous treatment with LG283, although other transcription factors, ZEB1 and ZEB2, did not change in their mRNA expression (Fig. 2c). Similarly, protein levels of SNAIL1 and 2 were increased by TGF-β2. However, the increase was reduced by simultaneous LG283 treatment (Fig. 2d). Protein levels of ZEB1 and 2 and TWIST1 were not significantly changed following treatment with TGF-β2 and/or LG283 at 96h (Fig. 2d). Western blotting exhibited the antagonizing effect of LG283 on TGF-β1-dependent p-Smad3 expression (Fig. 2e). Thus, LG283 significantly blocks TGF-β-induced EMT via specific inhibition of Smad3 phosphorylation and subsequent Snail signaling in epithelial cells.
LG283 suppresses EndoMT in cultured HUVEC cells
EndoMT may affect microvascular derangement and loss of functional endothelial cells leading to poor capillary bed formation, impaired angiogenesis, and chronic tissue ischemia in addition to tissue fibrosis. Indeed, endothelial dysfunction is considered a crucial factor for peripheral vessel remodeling in SSc (18, 29-31).
We used human endothelial HUVEC cells to examine the effects of LG283 in an EndoMT assay. Upon treatment with a cocktail containing rTGF-β2, TNF-α and IL-1β, the cells exhibited reduced expression of CD31 mRNA, a representative endothelial marker, and increased expression of FN-1 mRNA, a representative mesenchymal marker, by 24 h (Fig. 3a). However, simultaneous treatment with LG283 resolved the disparate mRNA expression patterns of decreased endothelial marker CD31 and increased mesenchymal marker FN-1 in response to the cytokine cocktail by 24 h and 48h, respectively (Fig. 3a). Next, the mRNA expression levels of EndoMT-associated transcription factors were investigated in HUVEC cells treated with the cytokine cocktail and/or LG283 for 48 hours. Expression levels of both SNAIL1 and SNAIL2 were remarkably increased following stimulation with the cytokine cocktail; however, this increase was significantly inhibited by simultaneous treatment with LG283 (Fig. 3b). On the other hand, the expression of ZEB1 and ZEB2 mRNAs were almost unchanged following treatment with the cytokine cocktail and/or LG283. Therefore, LG283 appears to suppress the effect of cytokines, including TGF-β-induced EndoMT, via specific inhibition of Snail signaling in endothelial cells.
LG283 inhibits the development of bleomycin-induced skin fibrosis in mice
Using a bleomycin-induced skin fibrosis mouse model, we examined the in vivo antifibrotic effects of LG283. Subcutaneous bleomycin injection and oral LG283 were co-administrated daily for 4 weeks. No apparent side effects including the change of body weight and activity were observed in any mice (data not shown). Histologically, skin thickness was increased more than two-fold following bleomycin injection, which was significantly reduced by both doses (40 mg/kg and 80 mg/kg) of oral LG283 (Fig. 4a, upper columnsand b, left). Similarly, the Masson’s trichrome-stained area was significantly reduced in bleomycin-injected skin sections from LG283 treated-mice, compared to those from mice treated with placebo (Fig. 4a, lower columns and b, right).
LG283 suppresses the reduction of capillary vessels in skin of bleomycin-treated mice
To investigate the effect of LG283 on vascular injury, capillary vessels were stained with anti-CD31 antibody in bleomycin-injected skin on day 28. Subcutaneous bleomycin injection reduced the capillary vessels (Fig. 4c), similar to what is seen in the skin of SSc patients. However, simultaneous administration of oral LG283 significantly suppressed this decrease in capillary vessels in the skin (Fig. 4c). Thus, this suggests LG283 treatment is protective against destructive vascular injury during the process of skin fibrosis.
LG283 does not affect inflammatory cell infiltration during the early-stage of bleomycin-induced skin fibrosis
Subcutaneous injection of bleomycin induces an early and transient inflammation mediated by locally infiltrating macrophages and other inflammatory cells (6). Local injection of bleomycin, but not control saline, induces increased infiltration of F4/80-positive macrophages into the dermal and subcutaneous tissues at day 7 (Fig. 4d). In addition, there was evident local infiltration of CD3-positive T cells in bleomycin-injected skin, but not in control skin (p<0.05; Fig. 4d). However, oral LG283 administration did not affect the infiltration of these cell subsets.
To further characterize the macrophage subset present in bleomycin-treated skin, we isolated CD11b-positve leukocytes from the lesional skin on day 21 and stained for monocyte/macrophage surface markers. As reported previously (32), proinflammatory macrophages (CD11b+Ly6Chi) and profibrotic M2 macrophages (CD11b+CD204+) were both increased in bleomycin-injected skin. Oral LG283 did not significantly reduce the infiltration of macrophage subsets (Fig. 4e). Thus, LG283 does not appear to significantly affect the skin inflammation induced by bleomycin injection.
LG283 does not affect proinflammatory or profibrotic cytokine production in bleomycin-injected skin
The process of early inflammation and subsequent fibrosis following subcutaneous bleomycin injection can be associated with increased production of various proinflammatory and profibrotic cytokines. In general, the concentrations of investigated cytokines, IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α and interferon (IFN) -γ, were increased in fluid from bleomycin-injected skin at day 7 (Fig. 5a). Among these cytokines, the concentration of IL-10, a representative regulatory cytokine, in whole extracts from bleomycin-injected skin was significantly reduced by co-administration of oral LG283. However, oral LG283 treatment did not significantly change the concentration of proinflammatory cytokines, such as IL-2, IL-6, IL-17A, TNF-α and IFN-γ, or of a profibrotic cytokine, IL-4.
LG283 antagonizes the expression of phosphorylated Smad3 in bleomycin-injected skin
TGF-β/Smad signaling has been considered to be essential for tissue fibrosis. Therefore, the effect of LG283 treatment on TGF-β/Smad signaling was evaluated in the bleomycin-injected skin at day 7. The expression of TGF-β1 mRNA in bleomycin-injected skin was not significantly affected by oral LG283 (Fig. 5b). Similarly, LG283 administration did not change the concentration of TGF-β1 protein in the bleomycin-injected skin extraction fluid (Fig. 5c). Expression levels of Smad3 protein were not affected by bleomycin and/or LG283 treatment (Fig. 5d). In contrast, expression of p-Smad3 protein was markedly increased in bleomycin-injected skin compared to controls, an effect that was significantly inhibited by LG283 treatment of mice (Fig. 5d). Thus, LG283 treatment specifically inhibits the expression of the p-Smad3 in the skin of bleomycin-induced skin fibrosis model.
LG283 suppresses Snail expression in bleomycin-injected skin
Since our in vitro findings indicate that LG283 inhibits both TGF-β-induced EMT and EndoMT, we examined the in vivo expression of transcription factors associated with EMT and/or EndoMT. Similar to what was seen in vitro, expression of Snail1 and Snail2 mRNAs were significantly reduced in bleomycin-injected skin following administration of oral LG283 on day 7 (Fig. 6a). However, expression of Zeb1, Zeb2, and Twist1 mRNAs were not significantly changed by oral LG283.
Consistent with these findings, immunohistopathology showed that the expression of Snail1 and 2 were augmented in bleomycin-injected skin on day 7. However, oral LG283 inhibited the expression of both transcription factors in epidermal keratinocytes, follicular epithelial cells, and dermal fibroblasts (Fig. 6b). Immunofluorescent staining revealed induction of Snail1 expression in CD31-positive endothelial cells and F4/80-positive macrophages in the dermis following bleomycin injection, an effect suppressed in mice treated with oral LG283 (Fig. 7). Thus, LG283 treatment specifically inhibits expression of the Snail transcription factor in skin cells, including keratinocytes and endothelial cells, following bleomycin treatment.