BMP-4 treatment of C3H10T1/2 stem cells blocks expression of MMP-3 and MMP-13

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Abstract

Microarray gene expression profiling was used to identify bone morphogenetic protein-4 (BMP-4) responsive factors involved in late stages of adipocyte commitment in C3H10T1/2 cells. The analysis revealed that the matrix metalloproteinase-3 (MMP-3) gene decreased 100-fold after BMP-4 treatment, and expression of MMP-13 decreased 19.5-fold. Uncommitted C3H10T1/2 cells exhibit dramatic up-regulation of MMP-3 and MMP-13 genes as cells become confluent. Real-time RT-PCR demonstrated that BMP-4 blocks expression of both transcripts. Likewise, a stable committed preadipocyte line derived from C3H10T1/2 cells did not express MMP-3 or MMP-13 at confluence, despite never receiving BMP-4. Active forms of both proteins were detected in media from confluent C3H10T1/2 cells but not in BMP-4 treated cells. Addition of BMP-4 to confluent C3H10T1/2 cells repressed the expression of both genes but did not induce adipocyte differentiation. The findings indicate that BMP-4-induced down-regulation of MMP-3 and MMP-13 is associated with commitment, but is insufficient to induce adipogenesis.

Section snippets

Materials and methods

Cell culture. A33 and 3T3-L1 cells were propagated and differentiated as described [8], [26]. The 10T1/2 cell line (American Type Culture Collection, Manassas, VA) was maintained in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% calf serum, and antibiotics. To commit the cells to adipocytes, 10T1/2 cells were plated at a density of 8 × 104 cells/6-cm dish, and 50 ng/ml BMP-4 (R&D Systems, Minneapolis, MN) was added two days after plating and replenished two days later. Once the

Commitment by BMP-4 alters the expression of numerous transcripts in postconfluent 10T1/2 cells

10T1/2 stem cells were committed to the adipocyte lineage by treatment with BMP-4 and grown to postconfluence. When subjected to our standard differentiation protocol [26], the committed cells differentiated into adipocytes in a manner indistinguishable from that of 3T3-L1 preadipocytes. In contrast, 10T1/2 cells not committed with BMP-4 remain undifferentiated despite treatment with differentiation inducers [11]. The molecular basis of the commitment process, however, has not been elucidated.

Discussion

Limited information is available about the commitment process of the adipocyte development program. Treatment of 10T1/2 stem cells with a DNA methylation inhibitor enabled the Weintraub laboratory and others to discover a number of muscle-specific transcription factors that, when expressed in a multipotent cell line, commit those cells to the muscle lineage. However, an adipocyte-specific commitment factor has yet to be identified. Our laboratory has shown that 10T1/2 cells proliferated in the

Acknowledgments

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health Research Grant DK66627 (to M.D.L.) and National Research Service Award Grant DK074294 (to R.R.B.).

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