Transforming growth factor-beta (
TGF-beta) is a multifunctional
growth factor that plays a critical role in tissue repair and
fibrosis.
Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in
collagen gene regulation. The present study was undertaken to determine whether endogenous
sphingolipids are involved in the
TGF-beta signaling pathway.
TGF-beta treatment induced endogenous
ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I)
collagen promoter/reporter gene construct (
COL1A2),
C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and
TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous
sphingolipids on
TGF-beta signaling we employed ectopic expression of
enzymes involved in
sphingolipid metabolism.
Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal
COL1A2 promoter activity and cooperated with
TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and
COL1A2 mRNA levels. Conversely, overexpression of
sphingosine kinase (SPHK1) inhibited basal and
TGF-beta-stimulated
COL1A2 promoter activity. These results suggest that endogenous
ceramide, but not
sphingosine or
sphingosine 1-phosphate, is a positive regulator of
collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2.
TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of
COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous
sphingolipid mediators in regulating the
TGF-beta signaling pathway.