PARENT SESSION
GROWTH FACTORS

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(669) DIFFERENTIAL BINDING AND NEUTRALIZATION OF TGF FAMILY LIGANDS BY FOLLISTATIN ISOFORMS.

Sidis, Yisrael¹, Mukherjee, Abir¹, Schneyer, Alan¹, ¹ Massachusetts General Hospital, Boston, MA

ABSTRACT- Follistatin (FS), a secreted monomeric glycoprotein expressed in a variety of tissues including pituitary and gonads, regulates activin mediated cell growth, differentiation, and secretion. FS-null mice die shortly after birth, and granulosa cell-specific gene inactivation causes fertility defects. FS is thought to function by binding and neutralizing activin and other TGF family members. Multiple FS isoforms, resulting from alternate splicing (FS315 and FS288) and posttranslational proteolytic modification (FS299-FS303), coexist in vivo. The present studies were undertaken to evaluate the capacity of the FS isoforms to regulate TGF family ligand activity, when FS acts in endocrine versus auto/paracrine mode. We first produced highly purified recombinant, tagged hFS288, 303 and 315 and evaluated their affinities for activin A, myostatin and BMP7 using heterologous competition assays with activin A as the trace. Binding to both activin and myostatin was similar among the isoforms (ED50s = 8 and 16 ng/well, respectively). Binding of FS to BMP7 was weaker, but significantly higher for FS288 (200 vs. 800ng/well). The capacity of FS isoforms to neutralize the biological activity of TGF family ligands was evaluated using reporter assays in HepG2 or HEK293 cells. While FS isoforms, when added exogenously, did not differ significantly in their ability to repress activin and myostatin signaling, FS288 was more potent (2-5 fold) when FS isoforms were expressed endogenously. BMP7 activity was impacted most by FS288, reflecting its higher affinity for BMP7 compared to the other FS isoforms. None of the isoforms had any effect on BMP4 or BMP2 signaling. To establish a possible link between function and subcellular localization of FS isoforms we performed immunofluoresence analysis. In transfected CHO cells, FS315 showed distinct punctuate distribution suggesting its presence in the Golgi/secretory pathway of the cell while FS288 was more diffuse throughout the cell. FS303 distribution pattern was intermediate but similar to that of FS315. Taken together, these data indicate that activin and myostatin are the preferable ligands of FS in circulation, and that all 3 FS isoforms have similar activity when acting in endocrine mode while FS288 is more potent as an auto/paracrine agent.

KEY WORDS: activin, TGFbeta, follistatin, myostatin