Endocrine Abstracts

Introduction: Hypoxia is involved with various physiological activities from glucose metabolism to tumor suppression. Hypoxia-inducible factor (HIF) is known as master regulator of oxygen homeostasis that triggers more than 1,000 related gene expressions. When hypoxia occurs, HIF1a protein is stabilized and starts transcription. Mucin1 mediates the stabilization of HIF1a in a cytoplasm. In addition, cytoplasmic domain of mucin1 and HIf1a form a transcriptional complex at glycolytic gene promoters. Interestingly, in calbindin-d9k knock out mice, both HIF1a and mucin1 were up-regulated in protein level. Calbindin-d9k has been known as a cytosolic calcium-binding protein. However, our results suggest that calbindin-d9k is involved with the interaction between HIF1a and mucin1. In the result of western blots, immunofluorescent and immunoprecipitation, calbindin-d9k was identified to interact with mucin1, which influence the stabilization of HIF1a.

Materials and methods: Eight weeks old C57BL/6 mice and calbindin-d9K Knockout mice were exposed to hypoxia for 3 weeks. Hypoxic condition was created in polycarbonate chamber with nitrogen supply to remove oxygen. Oxygen concentration were measured and maintained thoroughly about 12±2% partial pressure of O2. Expression of HIF1a and mucin1 protein in kidney were analyzed by Western blotting. Tissue-specific localization of calbindin-d9k and mucin1 were identified by immunofluorescent in kidney. Co-immunoprecipitation was performed to detect calbindin-d9k and mucin1 protein complex.

Results: In the result of western blot, expression of HIF1a and mucin1 were upregulated in calbindin-d9k knockout mice compared to that of wild type mice exposed in normal atmosphere. However, in hypoxia, both knockout and wild type mice showed similar protein expression. Cabindin-d9k and mucin1 were simultaneously detected at the distal convoluted tubules observed by immunofluorescent. In addition, protein complex between calbindin-d9k and mucin1 was identified by co-immunoprecipitation.

Conclusions: Calbindin-d9k was newly identified to interact with mucin1 protein in kidney. Upregulation of HIF1a protein in calbindin-d9k knock out mice might result from the absence of calbindin and mucin interaction.