Smad7 selectively interferes with different pathways of activin signaling and inhibits erythroid leukemia cell differentiation.

Smad family proteins are essential for transforming growth factor beta (TGF-beta) signal mediation downstream of a heteromeric complex of the type I and type II receptor serine/threonine kinases. A distant family member, Smad7, is expressed in most mammalian tissues and cells and prevents TGF-beta signaling. In this study, we examined the physiologic role of Smad7 in mediating the effects of activin, a member of the TGF-beta superfamily of peptides that functions in a number of processes, including blood-cell development. We report here that Smad7 expression is specifically absent in particular hematopoietic cells that respond to activin by differentiating into the erythroid lineage and that ectopic production of Smad7 causes mouse erythroid leukemia (F5-5) cells to become resistant to activin induction of erythroid differentiation. When coexpressed with type I activin receptor ActR-I or ActR-IB in concert with type II receptor ActR-II, Smad7 efficiently reduced an early transcriptional response mediated by ActR-I but had only a minimal effect on the response mediated by ActR-IB. In the presence of Smad7, overexpression of an activated form of ActR-IB, but not of an activated form of ActR-I, induced F5-5 cells to differentiate. These results suggest that Smad7 selectively interferes with the ActR-I pathway in activin signal transduction. The findings also indicate the existence of a novel activity of Smad7 that inhibits erythroid differentiation by blocking intracellular signaling of activin.

Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit.

The high affinity interaction of integrin alpha5beta1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on alpha5beta1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-alpha5 mAbs (P1D6 and JBS5) to alpha5beta1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to alpha5beta1. In contrast, binding of the anti-beta1 mAb P4C10 to alpha5beta1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with alpha5beta1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the alpha5 subunit. Our results indicate that the synergy region is recognized primarily by the alpha5 subunit (in particular by its NH2-terminal repeats) but that the beta1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.