LAUSR

Hypoxia has been implicated as a causal factor in mediating adipocyte dysfunction in obesity. Moreover, protein kinase D 1 (PKD1), a serine/threonine protein kinase, has been shown to contribute to diet-induced adiposity. Therefore, we investigated if PKD isoforms mediate hypoxia-induced dysfunction in 3T3-L1 adipocytes. Hypoxia increased phosphorylation of PKD1 at serine 916 (S916), the autophosphorylation site linked to PKD1 activation, indicating hypoxia-induced activation of PKD1 in adipocytes. Inhibition or depletion of PKD isoforms mitigated hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), the master transcription factor mediating hypoxia-induced gene expression, confirming that PKDs modulate the hypoxia-induced mechanism in adipocytes. Surprisingly, depletion of PKD1 and PKD2, but not PKD3, attenuated hypoxia-induced HIF1α target gene expression. Unlike PKD3, PKD1 and PKD2 possess a unique PDZ-binding motif at their C-terminus. Indeed, hypoxia upregulated a PDZ-containing scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) and its interaction with PKD1, whereas NHERF1 depletion attenuated hypoxia-induced PKD1 phosphorylation, HIF1α protein accumulation, and gene expression. Mechanistically, hypoxia induced nuclear import of active PKD1, which phosphorylated histone deacetylase 5 (HDAC5) at S498 that promoted cytoplasmic localization of HDAC5. HDAC5 deacetylated heat shock protein 70 (HSP70) at lysine 77, which dissociated HSP70 from HIF1α, allowing HSP90 association that stabilized HIF1α. Interestingly, PKD inhibition reversed hypoxia effects on subcellular localization of PKD1/HDAC5, HSP70 acetylation, and HIF1α/HSP90 association. In summary, our findings reveal a NHERF1-PKD1-HDAC5 mechanism modulating hypoxia-induced gene expression in adipocytes.