The synthesis of an abundant extracellular matrix (ECM), together with a harmful microenvironment triggers an evolutionary conserved mechanism known as the unfolded protein response (UPR). Activation of UPR allows an endoplasmic reticulum (ER) homeostasis leading to cell survival and appropriate ECM synthesis. If sustained, activation of the UPR leads to apoptosis. Recently, we have shown that PiT1, mostly described as a phosphate transporter, is expressed in the ER of chondrocytes. To elucidate its cellular functions and physiological role, we have generated inducible chondrocyte-specific PiT1 knockout mice (PiT1cKO) by crossing PiT1lox/lox with Agc1tm(IRES-creERT2 mice and treated by tamoxifen at postnatal day (P) 3 (french ethical approval #02286.01). Histological analysis of humerus sections at P5 revealed the presence of a hypocellular zone in the center of the growth plate due to massive cell death. Analysis of the phenotype at earlier stages showed an upregulation of the UPR-associated pro-apoptotic factor CHOP in PiT1-depleted cells. The ultrastructure of PiT1cKO chondrocytes analysed by electron microscopy showed a massively distended ER, a hallmark feature of ER-stressed cells. In vitro, triggering of an ER stress in primary chondrocytes and ATDC5 chondrogenic cells led to a strong induction of PiT1 expression which was dependent on the UPR transducers ATF6N, ATF4 and XPB1s. Furthermore, PiT1-depleted ATDC5 cells showed an increased sensitivity to ER stress-induced apoptosis as demonstrated by CHOP upregulation. Importantly, overexpression of the wild-type PiT1 or phosphate transport-deficient PiT1 mutant rescued this phenotype, illustrating that the implication of PiT1 in UPR is independent from its phosphate transport function. Our results suggest that under ER stress conditions, PiT1 regulates balance between chondrocyte survival and death. Of importance, the phenotype of PiT1cKO mice is reminiscent of HiF1α and PTEN deletion in cartilage. The mechanistic links between HiF1α/ PTEN pathways and PiT1 are currently being investigated in our lab.
Disclosure: The authors declared no competing interests. This work was supported by Région Pays de la Loire (Nouvelles Equipes nouvelles Thématiques grant). Greig Couasnay was the recipient of a Région Pays de la Loire doctoral fellowship.