Caveolin-1 increases basal and TGF-β1-induced expression of type I procollagen through PI-3 kinase/Akt/mTOR pathway in human dermal fibroblasts
Introduction
Caveolae are 50- to 100-nm sized, omega-shaped plasma membrane invaginated pits [1] that contain numerous small lipid patches enriched with cholesterol and glycosphingolipids [2], [3], [4]. Caveolae are considered to serve as platforms for dynamic association of signaling proteins and for the initiation or modulation of signaling [5], [6], [7]. The inner surface of caveolae is coated with scaffolding protein formed by members of the caveolin family (caveolin-1, -2, and -3) [2], [8]. Caveolins are highly hydrophobic proteins with a characteristic hairpin shape; caveolins are located within the plasma membrane, with both ends facing the cytoplasm. The N-terminal domain of the protein mediates homo- and hetero-oligomerization of the caveolin monomers as well as its binding to different molecules [9], [10], [11], [12]. Caveolin-1 plays a regulatory role in signaling as a direct inhibitor of a variety of plasma membrane-initiated signaling cascades including those of TGF-β, EGFR, and PKC [3], [13].
Type I collagen, the major structural component of the extracellular matrix, is a heterotrimer composed of three alpha chains encoded by the COL1A1 and COL1A2 genes [14]. Type I collagen comprises approximately 84% of the collagen synthesized by fibroblasts, and large depositions of type I collagen lead to skin and internal organ fibrosis [15]. TGF-β is known to regulate extracellular matrix (ECM) metabolism and the genesis of tissue fibrosis through overproduction of type I collagen [16], [17], [18], [19]. Several signaling pathways, including smad and ERK pathways, have been implicated in mediating TGF-β-induced extracellular matrix production and fibrosis [20], [21], [22]. However, despite recent research to understand the regulation of collagen expression, the regulatory mechanism of collagen gene expression has not been fully elucidated in human dermal fibroblasts.
The aim of this study was to investigate the role of caveolin-1 in type I procollagen expression in human dermal fibroblasts. In this study, we found that basal and TGF-β1-induced expression of type I procollagen are regulated by caveolin-1, which inhibits TGF-β1/smad signaling and activates PI-3 Kinase/Akt/mTOR pathways in human dermal fibroblasts, ultimately resulting in increased type I procollagen expression.
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Reagents
Dulbecco's modified Eagle's medium (DMEM) and antibiotics were purchased from Life Technologies, (Rockville, MA). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). Rabbit polyclonal anti-p-Akt and anti-p-smad3 were purchased from Cell Signaling Technology (Beverly, MA). The mouse monoclonal anti-type I procollagen antibody, SP1.D8, was used. LY294002 was purchased from Calbiochem (San Diego, CA). Rapamycin was obtained from Biomol (Plymouth Meeting, PA).
Cell cultures
Primary human dermal
Basal expression of type I procollagen is increased by cav-1 overexpression, but decreased by cav-1 siRNA in human dermal fibroblasts
To verify the role of cav-1 in type I procollagen expression, human dermal fibroblasts were infected with adenovirus expressing either GFP (Ad-GFP) only or human cav-1 cDNA (Ad-cav-1) for 24 h and further cultured in serum-free media for 72 h. Type I procollagen and cav-1 expression were measured by Western blotting of both culture media and whole cell lysates. Our results showed that type I procollagen expression was dose-dependently increased by Ad-cav-1 (Fig. 1A). Infection of 25 and 50 MOI
Discussion
In this study, we found that cav-1 significantly increased the basal and TGF-β1-induced type I procollagen expression, even though cav-1 overexpression inhibited TGF-β1/smad signaling. Previously, it was reported that cav-1 inhibited TGF-β/smad signaling through an interaction with the TGF-β type I receptor [13]. Our results also demonstrated that TGF-β1-induced smad3 phosphorylation and TGF-β1-induced transcriptional activities were significantly prevented by cav-1 overexpression. In addition
Acknowledgement
This research was supported by a grant (R11-2002-097-06001-0) through the Center for Aging and Apoptosis Research at Seoul National University from the Korean Science & Engineering Foundation (KOSEF) and by a research agreement with Amorepacific R&D Center.
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Present address: Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-gu, Seoul 135-710.