Effects of suppressing Smads expression on wound healing in Hyriopsis cumingii

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-β/Smad signal transduction pathways are involved in all phases of wound healing. We have previously reported on two cytoplasmic signal transduction factors, SMAD3 and SMAD5 in mussel H. cumingii (named HcSmads), suggesting their involvements in wound healing. Here, SMAD4 was cloned and described. The full length cDNA of HcSmad4 was 2543 bp encoded 515 Amino acids. Deduced HcSmad4 protein possessed conserved MH1 and MH2 domains, nuclear location signals (NLS), nuclear exput signals (NES) and Smad activation domain (SAD). Transcripts of SMAD3, 4 and 5 were constitutively expressed in all detected tissues, at highest levels in muscles. Furthermore, HcSmad4 mRNA levels were significantly increased at incision site post wounding, and expression of downstream target genes of SMAD4, such as HcMMP1, HcMMP19, HcTIMP1 and HcTIMP2 were upregulated to a certain extent. Whatever knocked down HcSmad3/4 or treated by specific inhibitors of Smad 3 (SIS3), expression levels of these genes displayed a significantly downregulated tendency compared with the wound group. In addition, histological evaluation suggested that SMAD3 knockdown or SIS3 treatment was accelerated wound healing, and then SMAD4 knockdown delayed the process of wound healing in mussels. These data implicate that SMAD3/4 play an important role in tissue repair in mollusks.