Characterization of a novel prolyl hydroxylase 2 gene from mud crab Scylla paramamosain: Insights into its role in the regulation of hypoxia-inducible factor-1α

Prolyl hydroxylase 2 (PHD2) is the key oxygen sensor that regulates the stability of the hypoxia-inducible factor -1 alpha (HIF-1 alpha). In this study, a novel PHD2 gene from the mud crab Scylla paramamosain, named SpPHD2, was cloned and identified. The full-length transcript of SpPHD2 was found to be 1926 bp, consisting of a 333 bp 5' untranslated region, a 1239 bp open reading frame, and a 354 bp 3' untranslated region. The putative SpPHD2 protein contained a Prolyl 4-hydroxylase alpha subunit homologues (P4Hc) domain in the C-terminal and a Myeloid translocation protein 8, Nervy, and DEAF-1 (MYND)-type zinc finger (zf-MYND) domain in the N-terminal. The mRNA expression of SpPHD2 was found to be widely distributed across all examined tissues. Additionally, the subcellular localization results indicated that the SpPHD2 protein was mainly localized in the cytoplasm. The in vivo silencing of SpPHD2 resulted in the upregulation of SpHIF-1 alpha and a series of downstream genes involved in the HIF-1 pathway, while SpPHD2 overexpression in vitro dose-dependently reduced SpHIF-1 alpha transcriptional activity, indicating that SpPHD2 plays a crucial role in SpHIF-1 alpha regulation. Interestingly, the expression of SpPHD2 increased under hypoxic conditions, which was further inhibited by SpHIF-1 alpha interference. Moreover, four hypoxia response elements were identified in the SpPHD2 promoter, suggesting that a feedback loop exists between SpPHD2 and SpHIF-1 alpha under hypoxia. Taken together, these results provided new insights into the regulation of SpPHD2 in response to hypoxia in S. paramamosain.