Inorganic phosphate modulates responsiveness to 24,25(OH)₂D₃ in chondrogenic ATDC5 cells

Abstract

Chondrogenic ATDC5 cells were used as a model of in vitro endochondral maturation to study the role of inorganic phosphate (Pi) in the regulation of growth plate chondrocytes by vitamin D3 metabolites. ATDC5 cells that were cultured for 10 days post-confluence in differentiation media and then treated for 24 h with Pi produced a type II collagen matrix based on immunohistochemistry and expressed mRNAs for several chondrocytic markers, including aggrecan, collagen types II and X, cartilage oligomeric matrix protein, and SOX9. Pi also caused a decrease in [(35)S]-sulfate incorporation and stimulated apoptosis, as evidenced by increased DNA fragmentation and caspase-3 activity. In addition, treatment with Pi induced sensitivity to 24,25-dihydroxyvitamin D3 and this effect was both dose-dependent and was blocked by phosphonoformic acid (PFA), a specific inhibitor of sodium dependent type III Pi transporters. Treatment with 24R,25(OH)(2)D(3) reduced cell number and increased alkaline phosphatase specific activity in a dose-dependent manner. Moreover, 24R,25(OH)(2)D(3) reversed the Pi-induced decrease in incorporation of [(3)H]-thymidine and [(35)S]-sulfate incorporation, as well as the Pi-induced increase in apoptosis. These results suggest that Pi acts as an early chondrogenic differentiation factor, inducing response to 24R,25(OH)(2)D(3); treatment of committed chondrocytes with Pi induces apoptosis, but 24R,25(OH)(2)D(3) mitigates these effects, indicating a possible inhibitory feedback loop.

The Inorganic Pyrophosphate Transporter ANK Preserves the Differentiated Phenotype of Articular Chondrocyte

“…Very few data are available on how ePP i could regulate gene expression in chondrocytes. In contrast, several evidences support an important role for inorganic phosphate (P i ) as an early differentiation factor in the ATDC5 cell line (43) and other mineralizing cells (44,45). However, mineralizing cells have a high TNAP activity which was not the case of our mature chondrocytes.…”

24R,25-Dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1α,25(OH)2D3 in hypertrophic cells

“…We have also observed Pi-induced apoptosis in ATDC5 cells, a mouse chondrogenic cell line that exhibits a resting zone chondrocyte-like phenotype as evidenced by expression of collagens II and X, Sox9, and cartilage oligomeric matrix protein (COMP) [2,29]. ATDC5 cells respond to 24R,25(OH) 2 D 3 with increased alkaline phosphatase activity and decreased cell number [29].…”

Membrane Estrogen Signaling Enhances Tumorigenesis and Metastatic Potential of Breast Cancer Cells via Estrogen Receptor-α36 (ERα36)

“…Confluent cultures of HCC38 cells were cultured in the presence of 0.1, 1.0, and 10 M chelerythrine for 24 h, and MTT was measured. In addition, HCC38 cells were treated with 5 and 7.5 mM phosphate to induce apoptosis, as we have previously shown that phosphate induces apoptosis in other cell types (24). We previously showed that tamoxifen, an ER agonist that inhibits PKC, blocks the stimulatory effects of E 2 on proliferation.…”