Abstract

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

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