The transforming growth factor-␤ (TGF-␤) maintains epithelial homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-␤ promotes malignancy. TGF-␤ activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-␤-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-␤ receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-␤-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-␤ on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses.

Smad7 is an inhibitor of the transforming growth factor-β-activated signaling pathway. Under welloxygenated conditions, Smad7 is a potent inhibitor of carcinoma cell invasion. Paradoxically, however, the expression of Smad7 is upregulated across several cancers and may promote cancer progression. Hypoxia, which is frequently met in solid tumors, is an enhancer of carcinoma cell invasion and cancer progression. Here, we report that hypoxia activates the expression of Smad7 in a hypoxia-inducible factor-and von HippelLindau protein-dependent manner. As expected, in normoxia, the forced expression of Smad7 inhibited carcinoma cell invasion. In contrast with the normoxic condition, the inhibitory effect of Smad7 was lost under hypoxia. The block in carcinoma cell invasion by forced expression of Smad7 was released by hypoxia in two invasive carcinoma cell lines. Moreover, the noninvasive HaCaT keratinocytes become invasive upon simultaneous hypoxia and transforming growth factor-β stimulus. The hypoxia-activated invasion was attenuated by inhibiting Smad7 expression by short interfering RNA. Finally, the increased Smad7 expression in human carcinomas correlated with hypoxic gene expression. The data provide evidence that hypoxia could convert Smad7 function from an invasion inhibitor into an activator of invasion. Furthermore, they might shed light as to why increased Smad7 expression is detected in cancers. Cancer Res; 70(14); 5984-93. ©2010 AACR.