Both E2 and TGFβ1 are central to ECM production in the skin; however, little is known about the cellular mechanisms underlying E2-induced dermal fibrosis or the transcriptional and translational regulation of TGFβ. We are the first to report that, in the skin, E2 induces TGFβ1 and TGFβ2, and they are regulated through the MAPK pathway and EGR1. TGFβ1 and TGFβ2, in turn, induce Col22A1. Based on these observations, we propose a cellular mechanism to describe aspects of E2-induced dermal fibrosis.
Col22A1 has been identified in patients with SSc and is known to contribute to fibrosis. Whole exome sequencing of patients with diffuse cutaneous SSc showed an enrichment of genes in the ECM pathway, including Col22A1 (35). Additionally, whole exome sequencing of African American patients with SSc identified Col22A1 as a rare variant that may increase African American susceptibility to SSc (36). To characterize the regulation of Col22A1, our group reported that primary human dermal fibroblasts from patients with SSc and healthy controls release Col22A1 in direct response to TGFβ1 stimulation. We also showed that Col22A1 mediates the transition of fibroblasts to myofibroblasts (34). These data indicate that Col22A1 is detected in patients with SSc and likely promotes fibrosis. Our current study extends the understanding of Col22A1 induction by demonstrating that it can occur in direct response to a pro-fibrotic mediator that induces TGFβ, namely E2. Thus, E2 contributes to dermal fibrosis through inducing Col22A1.
We show that both TGFβ1 and TGFβ2 are induced by E2, as they are in a wound-healing model (22, 23), and both likely contribute to dermal fibrosis. Even though the TGFβ1 and TGFβ2 isoforms are encoded by different genes, both use the same signaling receptors and cascades (37–39) and likely contribute to E2-induced Col22A1 transcription.
This report also extends the number of E2-induced ECM proteins and mediators known to impact dermal fibrosis to include Col IA2, Col IIIA1, Col22A1, TGFβ1 and TGFβ2 in our ex vivo human skin organ culture model. Yet, Col IA2 and Col IIIA1 steady-state transcript levels were not increased in primary dermal fibroblast single-cell culture in vitro. The discrepancy between ex vivo and in vitro results may be explained by the inclusion of other cell types in ex vivo skin tissue, such as keratinocytes, which are responsive to E2 and can contribute to ECM formation (40–42).
While primary human dermal fibroblasts produce less Col IA1, Col IA2, FN and Col22A1 when pre-treated with the TGFβ receptor inhibitor SB-431542 before stimulation with TGFβ1 (34, 43), E2-induced Col IA2, Col IIIA1 and FN transcription was not prevented by pre-treatment with the inhibitor. This is likely because E2 can induce other pro-fibrotic mediators to influence ECM production. Our unpublished findings suggest that E2 increases the transcript and protein levels of the pro-fibrotic cytokine IL-6 (unpubished data), which can then increase collagen and FN levels through trans-signaling (44). IL-6 also activates STAT3, which promotes Col IA2 synthesis post-transcriptionally (45). Thus, E2-induced fibrosis likely occurs through both the non-canonical TGFβ-ERK and IL-6-STAT3 signaling pathways.
We investigated whether the MAPK pathway, which is central to E2-induced Col22A1 signaling, regulates TGFβ1 and TGFβ2. We report that E2-induced ERK1/2 phosphorylation in primary human dermal fibroblasts is vital to TGFβ1 and TGFβ2 expression, since blockade of the MAPK/ERK pathway was inhibitory. In apoptotic cells, TGFβ transcriptional and translational regulation also occurs through the MAPK pathway, in addition to the RhoA and PI-3K/AKT pathways (46). Specifically, TGFβ1 synthesis is induced by activation of CD36 on macrophages in response to apoptotic cells (47). Further studies are needed to elucidate the role of CD36 in E2-induced TGFβ1 and TGFβ2 transcription and translation in primary human dermal fibroblasts.
Transcription factors are crucial to comprehend E2-induced TGFβ1 and TGFβ2 transcription. EGR1 is a candidate transcription factor in this pathway because it has been reported downstream of TGFβ1 signaling in human dermal fibroblasts (30), and the EGR1-TGFβ1 relationship is important in a murine model of pulmonary fibrosis (48). We report that EGR1 is also upstream of E2-induced TGFβ1 transcription in primary human dermal fibroblasts, because EGR1 binds to and activates the TGFβ1 promoter (29). As confirmation, exogenous expression of EGR1 in the cancer cell line HT1080 led to increased secretion of TGFβ1 (49). Additionally, EGR1 increased E2-induced TGFβ2 transcription in primary dermal fibroblasts (50, 51).