Abstract
Prolyl 4-hydroxylases (P4Hs) have vital roles in regulating collagen synthesis and hypoxia response. A transmembrane P4H (P4H-TM) is a recently identified member of the family. Biallelic loss of function P4H-TM mutations cause a severe autosomal recessive intellectual disability syndrome in humans, but functions of P4H-TM are essentially unknown at cellular level. Our microarray data on P4h-tm−/− mouse cortexes where P4H-TM is abundantly expressed indicated expression changes in genes involved in calcium signaling and expression of several calcium sequestering ATPases was upregulated in P4h-tm−/− primary mouse astrocytes. Cytosolic and intraorganellar calcium imaging of P4h-tm−/− cells revealed that receptor-operated calcium entry (ROCE) and store-operated calcium entry (SOCE) and calcium re-uptake by mitochondria were compromised. HIF1, but not HIF2, was found to be a key mediator of the P4H-TM effect on calcium signaling. Furthermore, total internal reflection fluorescence (TIRF) imaging showed that calcium agonist-induced gliotransmission was attenuated in P4h-tm−/− astrocytes. This phenotype was accompanied by redistribution of mitochondria from distal processes to central parts of the cell body and decreased intracellular ATP content. Our data show that P4H-TM is a novel regulator of calcium dynamics and gliotransmission.
Highlights
The prolyl 4-hydroxylase (P4H) family of enzymes includes the collagen Prolyl 4-hydroxylases (P4Hs) and hypoxia-inducible factor (HIF) P4Hs that have vital roles in collagen synthesis and in the regulation of the hypoxia response, respectively (Myllyharju, 2008, 2013; Ratcliffe, 2013; Ivan and Kaelin, 2017)
Based on the GSEA analysis, we hypothesized that P4H-TM is involved in the regulation of active vesicular transport via calcium signaling in the brain. qRT-PCR analyses showed upregulation of P4H-TM mRNA expression over time in mouse cortical tissue from embryonic day (E)15 to one month of age (Fig. 1E)
The data show that the attenuation of receptor-operated calcium entry (ROCE) in P4h-tmÀ/À cells was reversed on treatment with siHIF1a, but not siHIF2a (Fig. 8G). These results indicate that the higher SERCA2 expression and reduced ROCE is mediated by stabilization of HIF1 in P4h-tmÀ/À astrocytes
Summary
The prolyl 4-hydroxylase (P4H) family of enzymes includes the collagen P4Hs and hypoxia-inducible factor (HIF) P4Hs that have vital roles in collagen synthesis and in the regulation of the hypoxia response, respectively (Myllyharju, 2008, 2013; Ratcliffe, 2013; Ivan and Kaelin, 2017). HIF-P4Hs hydroxylate two prolyl residues located in the oxygen-dependent degradation domain (ODDD) of the HIFa subunit in normoxia. Hydroxylation leads to von Hippel–Lindau (VHL)-targeted degradation of HIFa, which suppresses the transcription of hypoxia responsive genes in normoxia. Three members of the HIFP4H family were identified: HIF-P4H-1, HIF-P4H-2, and HIF-P4H-3 ( known as PHD1, PHD2, and PHD3 or EGLN2, EGLN1, and EGLN3; Myllyharju, 2013; Ratcliffe, 2013; Ivan and Kaelin, 2017)
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