Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation

Abstract

Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions.

Effects of matrix metalloproteinases on the fate of mesenchymal stem cells

“…At the protein level, the expression of MMP-3 and MMP-13 was significantly increased during adipogenic differentiation of MSCs with HTRA1. Moreover, MMP inhibitors (NNGH and CL-82198) for MMP-3 and MMP-13 during adipogenic differentiation increased the level of collagen type IV in HTRA1-treated MSCs, suggesting the important role of HTRA1 in inducing the expression of MMP-3 and MMP-13, and in adipogenic differentiation of MSCs [20]. Fibronectin was found, in vitro, to be produced first among all ECM components during adipogenesis.…”

Prostaglandin E2 inhibits matrix mineralization by human bone marrow stromal cell-derived osteoblasts via Epac-dependent cAMP signaling

“…Cell cultures were maintained at 37 °C, in 5% CO 2 and 98% humidity in normal growth medium consisting of Dulbecco’s modified eagle medium (DMEM-low glucose, with GlutaMAX) (Thermo Fisher Scientific, Reinach, Switzerland), supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), penicillin/streptomycin (50 units/ml; 50 μg/ml). Cells were used between passage 5 and 8 55 .…”

“…Cells were exposed to adipogenic induction medium for 3 days and subsequently maintained in IBMX-free adipogenic induction medium thereafter 55 . Triglyceride accumulation in hBMSCs undergoing adipogenesis was identified at specified time points using Oil Red O (Sigma-Aldrich), and staining quantified by measuring the optical densities of extracted stain at 510 nm.…”

“…Triglyceride accumulation in hBMSCs undergoing adipogenesis was identified at specified time points using Oil Red O (Sigma-Aldrich), and staining quantified by measuring the optical densities of extracted stain at 510 nm. Differences in Oil Red O staining due to cell proliferation were accounted for by normalization of optical densities to cell number as previously 55 .…”

“…Gene expression levels of osteogenic or adipogenic markers were quantified by RT-qPCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific) (Supplementary Table S1) as previously described 55 . Total RNA was harvested from cells at selected time points during differentiation and 0.5 μg of total RNA reverse-transcribed using Superscript II (Thermo Fisher Scientific).…”