Pathway In Human Dermal Fibroblasts Research Articles

Caveolin-1 increases basal and TGF-β1-induced expression of type I procollagen through PI-3 kinase/Akt/mTOR pathway in human dermal fibroblasts

Caveolin-1 (Cav-1) is a major structural protein of caveolae and plays an important role as a negative regulator of various signaling pathways such as the transforming growth factor-β (TGF-β)/smad pathway. In this study, we investigated the role of cav-1 on basal and TGF-β1-induced expression of type I procollagen in human dermal fibroblasts. Our results demonstrated that basal and TGF-β1-induced expression of type I procollagen were significantly increased by adenoviral cav-1 (Ad-cav-1) overexpression, while the basal level of type I procollagen was decreased by cav-1 siRNA. Overexpression of cav-1 inhibited TGF-β1-induced phosphorylation of smad3 and transcription of 3TP-Lux and SBE luciferase reporters, suggesting that cav-1 may inhibit the TGF-β1/smad signaling pathway. We observed that TGF-β1-induced type I procollagen expression was decreased by smad3 siRNA transfection. However, the reduction of TGF-β1-induced type I procollagen expression by smad3 siRNA was reversed by cav-1 overexpression. In addition, our results also showed that TGF-β1 treatment increased the phosphorylation of Akt, and Ad-cav-1 infection augmented this TGF-β1-induced phosphorylation of Akt. Ad-myr-Akt infection significantly increased the basal expression of type I procollagen. In contrast, TGF-β1-induced type I procollagen expression was decreased by Akt siRNA transfection and the PI3-kinase inhibitor, LY294002, inhibited the TGF-β1-induced type I procollagen expression and also inhibited the cav-1-induced expression of type I procollagen. In conclusion, our results suggest that cav-1 increases the basal and TGF-β1-induced expression of type I procollagen by regulating two opposite signaling pathways: inhibiting TGF-β1/smad signaling and activating a PI-3 kinase/Akt/mTOR-dependent pathway in human dermal fibroblasts, ultimately resulting in increased type I procollagen expression.

Ultraviolet B‐induced matrix metalloproteinase‐1 and ‐3 secretions are mediated via PTEN/Akt pathway in human dermal fibroblasts

Matrix Metalloproteinases (MMPs) are crucial enzymes for ultraviolet irradiation-induced photoaging in human skin. Ultraviolet B (UVB) stimulates dermal fibroblasts to increase MMP-1 and -3 expression and extracellular matrix (ECM) degradation in photoaging. We investigated whether phosphatase and tensin homolog (PTEN)/Akt pathway is involved in secretions of MMP-1 and -3 in human dermal fibroblasts. The increase in MMP-1 and -3 expression and secretion occurred along with the increase in PTEN and Akt phosphorylation by UVB irradiation in a dose- and time-dependent manner. However, treatment with a casein kinase 2 inhibitor, 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, inhibited their phosphorylations and MMP-1 and -3 secretions. Transfection of wild-type PTEN (Wt-PTEN) decreased basal and UVB-induced MMP-1 and -3 secretions, as well as activator protein-1 (AP-1) activity, while transfection of small interference RNA of PTEN (siRNA-PTEN), phosphatase-inactive PTEN (C124S-PTEN), or lipid phosphatase-inactive PTEN (G129E-PTEN) increased basal or UVB-induced MMP-1 and -3 secretions and AP-1 activity. Transfection of constitutively active Akt (Myr-Akt) also increased basal or UVB-induced MMP-1 and -3 secretions, as well as AP-1 activity. However, transfection of kinase-inactive Akt (K179M-Akt) decreased their secretions, but showed no significant change of AP-1 activity without UVB irradiation, and a significant increase of AP-1 activity with UVB irradiation. Treatment with the phosphatidylinositol 3-kinase inhibitors, LY294002 or wortmannin, downregulated basal and UVB-induced MMP-1 and -3 secretions. In conclusion, UVB irradiation increases PTEN and Akt phosphorylation in human dermal fibroblasts, and these inhibition of PTEN and activation of Akt by phosphorylation are involved in UVB-induced MMP-1 and -3 secretions partly through upregulation of AP-1 activity.

Selective inhibition of activin receptor–like kinase 5 signaling blocks profibrotic transforming growth factor β responses in skin fibroblasts

Members of the transforming growth factor beta (TGFbeta) cytokine superfamily play critical roles in both homeostasis and disease. In light of their profibrotic effects, these molecules are implicated in the pathogenesis of fibrosis. In fibroblasts, TGFbeta signals through the activin receptor-like kinase 5 (ALK-5) type I TGFbeta and triggers Smad and MAP kinase signaling pathways. Because targeting of TGFbeta signaling represents a potential approach to the treatment of systemic sclerosis (SSc) and other fibrotic disorders, we investigated the modulation of intracellular TGFbeta signal transduction by SB431542, the first small-molecule inhibitor of ALK-5 to be described. Ligand-induced activation of the Smad signaling pathway in human dermal fibroblasts was examined by Western blot analysis and confocal immunocytochemistry. Modulation of profibrotic gene expression was investigated using Northern blot analysis, transient transfection assays, and confocal microscopy. Induction of TGFbeta production was evaluated by enzyme-linked immunosorbent assay. SB431542 abrogated TGFbeta-induced phosphorylation and nuclear importation of endogenous Smad2/3 and Smad4, and inhibited Smad3- and Smad2-dependent gene transcription. Treatment with SB431542 prevented TGFbeta-induced stimulation of collagen, fibronectin, plasminogen activator inhibitor 1, and connective tissue growth factor gene expression, TGFbeta autoinduction, and myofibroblast transdifferentiation, and it could reverse stimulation even when added to the cultures after TGFbeta. In contrast, STAT-6-mediated stimulation of collagen gene expression induced by interleukin-13 was not prevented by SB431542, indicating the specificity of blockade for ALK-5-dependent signaling. Furthermore, in contrast to its effects on receptor-activated Smad activation, SB431542 failed to prevent TGFbeta-induced activation of MAP kinases. The results indicate that SB431542 is a potent inhibitor of intracellular TGFbeta signaling in normal fibroblasts through selective interference with ALK-5-mediated Smad activation and Smad-dependent transcriptional responses. Therefore, SB431542 is useful as a novel experimental tool for gaining a detailed understanding of normal and aberrant TGFbeta signaling in SSc. Furthermore, as an anti-TGFbeta agent, SB431542 may represent a potential new approach to the treatment of fibrosis.