SPARC Inhibits Adipogenesis by Its Enhancement of β-Catenin Signaling

Jing Nie,E Helene Sage

doi:10.1074/jbc.m808285200

Abstract

SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of beta-catenin and subsequently enhanced the interaction of beta-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on beta-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of alpha5 integrin was enhanced, and that of alpha6 integrin was inhibited. Lithium chloride, which enhances the accumulation of beta-catenin, also inhibited the expression of alpha6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation.

Highlights

The cellular composition of WAT includes primarily adipocytes and preadipocytes as well as endothelial cells and macrophages
We examined the effect of SPARC on the expression and activity of ␣5 and ␣6 integrin subunits during adipocyte differentiation. ␣5 was decreased during adipocyte differentiation (Fig. 11A), consistent with the report that overexpression of the ␣5 integrin subunit significantly inhibits adipogenesis [39]
Primary preadipocytes from SPARCnull mice exhibit an enhanced capacity for adipocyte differentiation and a diminished propensity toward osteoblast differentiation

Summary

EXPERIMENTAL PROCEDURES

Mice—A colony of WT and SPARC-null mice has been described [21]. C57BL/6 WT and SPARC-null mice are maintained in a specific pathogen-free facility. Cell culture reagents, including Dulbecco’s modified Eagle’s medium (DMEM), DMEM/F-12, trypsin/EDTA, antibiotics, and fetal bovine serum were purchased from Invitrogen. Isolation and Differentiation of Preadipocytes from Adipose Tissue—Stromal-vascular cells (SVCs) from mouse adipose tissue were prepared as described [25]. After washes with water to remove unbound dye, cells were photographed under a Leica inverted microscope equipped with a digital camera. Alkaline Phosphatase Staining—After 2 washes with phosphate-buffered saline, cells were fixed in methanol/acetone (1:1) for 10 min at Ϫ20 °C. The cells were subsequently washed twice with water and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium alkaline phosphatase substrate buffer (Sigma) for 45 min at 37 °C. The cells were scraped into Nuclei EZ lysis buffer and were pelleted by centrifugation after two washes with ice-cold serum-free DMEM. With a lysis buffer (1% Nonidet P-40, 0.1% SDS, 10 mM Tris-HCl, pH 7.5, and 5 mM EDTA) containing a com-

Ab lected by scraping with a policeman

RESULTS

Preadipocytes and mesenchymal stem cells differentiate into

DISCUSSION

GPDH activity

FN cell