BMP-2 induces the expression of activin betaA and follistatin in vitro

Activins are members of the transforming growth factor beta (TGF-beta) superfamily and have been shown to be multifunctional regulators of development and cell differentiation. Increasing evidence suggests activin betaA is involved in skeletal development. Using differential display PCR we have identified activin betaA as a gene associated with recombinant human bone morphogenetic protein-2 (rhBMP-2) induced differentiation of a mouse limb bud cell line, MLB13MYC clone 17, from a prechondroblastic to an osteoblastic phenotype. The expression of activin betaA peaks at 24 h of rhBMP-2 treatment, before detection of osteocalcin mRNA expression. Cycloheximide treatment inhibits induction of activin betaA, indicating a requirement for new protein synthesis. The induction of the mRNA encoding follistatin, an activin binding protein, was also examined. Follistatin mRNA increases within 18 h of rhBMP-2 treatment, as activin betaA mRNA increases but before it peaks. Treatment of MLB13MYC clone 17 cells with purified activin betaA concomitant with rhBMP-2 does not affect markers of chondrocyte or osteoblast differentiation, nor does treatment with purified activin betaA alone. This suggests that activin betaA exerts its effect via a paracrine mechanism. In situ hybridization analysis demonstrates that activin betaA expression is localized to cells in the developing interphalangeal joints of embryonic mouse limbs. This is consistent with in vivo induction by BMP-2 which is also expressed in the developing joints. Activin betaA, therefore, is downstream from BMP-2 in the cascade of events that result in skeletal development.