Phosphate-induced Apoptosis of Hypertrophic Chondrocytes Is Associated with a Decrease in Mitochondrial Membrane Potential and Is Dependent upon Erk1/2 Phosphorylation

Abstract

Growth plate abnormalities, associated with impaired hypertrophic chondrocyte apoptosis, are observed in humans and animals with abnormalities of vitamin D action and renal phosphate reabsorption. Low circulating phosphate levels impair hypertrophic chondrocyte apoptosis, whereas treatment of these cells with phosphate activates the mitochondrial apoptotic pathway. Because phosphate-mediated apoptosis of chondrocytes is differentiation-dependent, studies were performed to identify factors that contribute to hypertrophic chondrocyte apoptosis. An increase in the percentage of cells with low mitochondrial membrane potential, evaluated by JC-1 fluorescence, was observed during hypertrophic differentiation of primary murine chondrocytes in culture. This percentage was further increased by treatment of hypertrophic, but not proliferative, chondrocytes with phosphate. Phosphate-mediated apoptosis was observed as early as 30 min post-treatment and was dependent upon Erk1/2 phosphorylation. Inhibition of Erk1/2 phosphorylation in vivo confirmed an important role for this signaling pathway in regulating hypertrophic chondrocyte apoptosis in growing mice. Murine embryonic metatarsals cultured under phosphate-restricted conditions demonstrated a 2.5-fold increase in parathyroid hormone-related protein mRNA expression accompanied by a marked attenuation in phospho-Erk immunoreactivity in hypertrophic chondrocytes. Thus, these investigations point to an important role for phosphate in regulating mitochondrial membrane potential in hypertrophic chondrocytes and growth plate maturation by the parathyroid hormone-related protein signaling pathway.Maturation of the growth of long bones is dependent upon the differentiation of proliferative chondrocytes into prehypertrophic and subsequently hypertrophic chondrocytes. Terminal differentiation of hypertrophic chondrocytes is characterized by the expression of signaling molecules that promote vascular invasion, apoptosis, and replacement of hypertrophic chondrocytes by osteoblasts that lay down the primary spongiosa of bone. Aberrant regulation of this developmental process results in growth plate disorders. Although calcium has been shown to play an important role in regulating chondrocyte maturation (1), apoptosis of terminally differentiated hypertrophic chondrocytes is dependent upon normal levels of circulating phosphate (2).Rickets is a growth plate anomaly observed in growing animals and humans with abnormalities of vitamin D action and renal phosphate reabsorption (3-6). In vivo investigations in genetically modified and dietary-manipulated mouse models demonstrate that hypophosphatemia is the underlying metabolic abnormality that impairs growth plate maturation in these disorders: low circulating phosphate levels result in impaired apoptosis of terminally differentiated hypertrophic chondrocytes in the growth plate, leading to rickets (2). The observation that inhibition of phosphate transport prevents phosphate-mediated apoptosis in hypertrophic chondrocytes (7-9) further ...

Early Skeletal and Biochemical Alterations in Pediatric Chronic Kidney Disease

“…Acidemia, a condition associated with defective skeletal mineralization (17), was present in a substantial percentage of study participants; however, defective mineralization was equally prevalent in nonacidemic individuals, suggesting that acidemia did not affect the prevalence of abnormal mineralization in the current cohort. Moreover, levels of Consistent with the stimulatory actions of PTH on osteoblasts, circulating PTH levels were associated with increased osteoid accumulation in this study and, consistent with data from Hyp mice (35), phosphorus concentrations were consistently, independently, and inversely associated with both osteoid accumulation and osteoid maturation time. These findings suggest that altered phosphorus sensing or metabolism may be involved in the pathogenesis of the defective skeletal mineralization in pediatric CKD.…”

Intracellular Modulation of Signaling Pathways by Annexin A6 Regulates Terminal Differentiation of Chondrocytes

“…Previous studies from our and other laboratories have shown that ascorbic acid and additional 1.5 mM P i stimulated hypertrophic, terminal, and mineralization events of growth plate and sternal chondrocytes (11,15). Other studies showed that extracellular P i is transported back into chondrocytes via the Na ϩ /P i co-transporter where it acts as a signaling molecule that activates various signaling pathways, including the ERK-MAPK signaling pathway (23). In this study, we show that ascorbic acid and additional 1.5 mM P i activate PKC␣ and ultimately ERK and p38 MAPK signaling pathways.…”

“…Co-immunostaining with antibodies specific for type X collagen (hypertrophic marker) revealed that hypertrophic chondrocytes were equally well transfected as non-hypertrophic chondrocytes (data not shown). Cells were cultured in the absence or presence of 50 g/ml ascorbic acid and additional 1.5 mM inorganic phosphate (P i ), a myristoylated protein kinase C inhibitor [20][21][22][23][24][25][26][27][28]; specific PKC␣ inhibitor; 10 M; Calbiochem), PD98059 (specific ERK inhibitor; 10 M; Millipore, Billerica, MA), or SB203580 (specific p38 inhibitor; 10 M; Millipore). After 6 days of culture, cells were stained for APase activity using alkaline phosphatase magenta immunohistochemical substrate solution (Sigma), or APase activity was determined in cell lysates using p-nitrophenyl phosphate as a substrate as described previously (11).…”

Sox9 Reprogrammed Dermal Fibroblasts Undergo Hypertrophic Differentiation In Vitro and Trigger Endochondral Ossification In Vivo

“…GFP immunostaining suggested presence of both donor (52.49% -2.57%) and host cells (47.51% -2.57%) in the cartilaginous region (NS, not significant, p = 0.18), while bone tissue was thought to be completely host derived (E, F). differentiation, chondrocytes activate an apoptosis program as a result of phosphate stimuli and hence contribute to cartilage remodeling during EO (Miedlich et al, 2010). Surprisingly, higher levels of Runx2 were detected in iChon Con cells when compared to iChon Ind , whereas no collagen type X expression was detected.…”

“…Despite absence of donor contribution to bone formation, we can conclude that iChon Ind cells are able to trigger EO. Indeed, the results that have been published on whether hypertrophic chondrocytes are able to transdifferentiate to osteoblasts during EO are conflicting (Miedlich et al, 2010;Roach, 1992). Moreover, it has previously been reported that hypertrophic chondrocytes activate an apoptosis program while expressing vascular endothelial growth factor to stimulate blood vessel ingrowth (Adams and Shapiro, 2002;Gerber et al, 1999).…”