Proprotein convertase PACE4 is down-regulated by the basic helix‒loop‒helix transcription factor hASH-1 and MASH-1

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Abstract
PACE4 is a mammalian subtilisin-like proprotein convertase that activates transforming growth factor (TGF)-β-related proteins such as bone morphogenetic protein 2 (BMP2), BMP4 and Nodal and exhibits a dynamic expression pattern during embryogenesis. We recently determined that the 1kb 5′-upstream region of the PACE4 gene contains 12 E-box (E1–E12) elements and that an E-box cluster (E4–E9) acts as a negative regulator [Tsuji, Yoshida, Hasegawa, Bando, Yoshida, Koide, Mori and Matsuda (1999) J. Biochem. (Tokyo) 126, 494–502]. It is known that the mammalian achaete–scute homologue 1 (MASH-1) binds specifically to an E-box (CACCTG) sequence in collaboration with E47, a ubiquitously expressed basic helix–loop–helix (bHLH) factor. To identify the roles of the bHLH factor and E-box elements in regulating PACE4 gene expression in neural development, we analysed the effects of human achaete–scute homologue 1 (hASH-1) on PACE4 gene expression with various neuroblastoma cell lines. The expressions of PACE4 and hASH-1 are correlated inversely in these cell lines. The overexpression of hASH-1 or MASH-1 causes a marked decrease in endogenous PACE4 gene expression but has no effect on the expression of other subtilisin-like proprotein convertases such as furin, PC5/6 and PC7/8. In contrast, other neural bHLH factors (MATH-1, MATH-2, neurogenin 1, neurogenin 2, neurogenin 3 and E47) did not affect PACE4 gene expression. Furthermore, an E-box cluster was a negative regulatory element for the promoter activity in NBL-S cells expressing hASH-1 at high level as determined by a luciferase assay. Binding of hASH-1 to the E-box cluster was confirmed by gel mobility-shift assay. In the present study we identified the PACE4 gene as one of the targets of hASH-1, which is a key factor in the initiation of neural differentiation. These results suggest that the alteration of PACE4 gene expression by hASH-1 causes rapid changes in the biological activities of TGF-β-related proteins via post-translational modification of these proteins.

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