The molecular and cellular mechanisms that maintain proper collagen homeostasis in healthy human skin and are responsible for the dysregulated collagen synthesis in scleroderma remain primarily unknown. This study demonstrates that Fli1 is a physiological negative regulator of collagen gene expression in dermal fibroblasts in vitro and in human skin in vivo.

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Endothelial microparticles (EMPs) are complex vesicular structures that originate from plasma membranes of activated or apoptotic endothelial cells. EMPs play a significant role in vascular function by altering the processes of inflammation, coagulation, and angiogenesis, and they are key players in the pathogenesis of several vascular diseases. Circulating EMPs are increased in many agerelated vascular diseases such as coronary artery disease, peripheral vascular disease, cerebral ischemia, and congestive heart failure. Their elevation in plasma has been considered as both a biomarker and bioactive effector of vascular damage and a target for vascular diseases. This review focuses on the pleiotropic roles of EMPs and the mechanisms that trigger their formation, particularly the involvement of decreased estrogen levels, thrombin, and PAI-1 as major factors that induce EMPs in age-related vascular diseases.

Biosynthesis of fibrillar collagen in the skin is precisely regulated to maintain proper tissue homeostasis; however, the molecular mechanisms involved in this process remain largely unknown. Transcription factor Fli1 has been shown to repress collagen synthesis in cultured dermal fibroblasts. This study investigated the role of Fli1 in regulation of collagen biosynthesis in mice skin in vivo using mice with the homozygous deletion of the C-terminal transcriptional activation (CTA) domain of the Fli1 gene (Fli1 ⌬CTA/⌬CTA ). Skin analyses of the Fli1 mutant mice revealed a significant upregulation of fibrillar collagen genes at mRNA level, as well as increased collagen content as measured by acetic acid extraction and hydroxyproline assays. In addition, collagen fibrils contained ultrastructural abnormalities including immature thin fibrils and very thick irregularly shaped fibrils, which correlated with the reduced levels of decorin, fibromodulin, and lumican. Fibroblasts cultured from the skin of Fli1 ⌬CTA/⌬CTA mice maintained elevated synthesis of collagen mRNA and protein. Additional experiments in cultured fibroblasts have revealed that although Fli1 ⌬CTA retains the ability to bind to the collagen promoter in vitro and in vivo, it no longer functions as transcriptional repressor. Together, these results establish Fli1 as a key regulator of the collagen homeostasis in the skin in vivo.Fibril-forming collagens are the major structural components of the dermis responsible for its characteristic strength and resiliency. In the skin collagen fibrils are composed mainly of collagen type I and smaller amounts of collagen types III and V (11). Although collagen type V represents only a minor component of the fibril, it plays a key regulatory role in the process of fibrillogenesis (41). During physiologic remodeling, coordinate synthesis of specific collagen chains is tightly regulated (29), while during fibrosis the fibrillar collagens are produced at increased levels (37). The first critical step in the collagen biosynthetic pathway occurs at the level of transcription. In the past few years, a number of cis-regulatory elements and cognate transcription factors involved in type I collagen gene regulation at the basal level and in response to cytokines have been characterized in in vitro studies (8,13,18,36). Subsequently, several of these response elements, including Sp1 and CBF/nuclear factor 1 binding sites, have been validated in vivo in a transgenic mouse model (34). The in vivo studies have also underscored the complexity of the transcription regulation of the collagen gene, which involves interactions between transcription factor complexes at the proximal promoter and the far upstream enhancer (34).Additional intracellular steps in collagen fibrillogenesis involve collagen folding and trimerization, which take place in the endoplasmic reticulum (3, 23). Prolyl-4 hydroxylase (P4H) and protein disulfide isomerase, which together form a P4H tetramer, catalyze formation of hydroxyproline, a critical step that fa...

Transforming growth factor-␤ (TGF-␤) 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-␤ signaling pathway. TGF-␤ treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a ␣2(I) collagen promoter/reporter gene construct (COL1A2), C 6 -ceramide (10 M) exerted a stimulatory effect on basal and TGF-␤-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-␤ 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-␤ 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-␤-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-␤-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-␤ signaling pathway.