PTH ameliorates acidosis-induced adverse effects in skeletal growth centers: The PTH–IGF-I axis

Abstract

Chronic metabolic acidosis (CMA) exerts profound adverse effects on bone metabolism thereby leading to impaired skeletal linear growth. We have recently shown that CMA in vitro causes distinct morphological changes in skeletal growth centers along with inhibition of endochondral differentiation. In addition, CMA causes an end organ resistance to the anabolic effects of growth hormone (GH) and locally produced insulin-like growth factor-I (IGF-I) in skeletal growth centers. Given the effects of parathyroid hormone (PTH) and PTH related protein (PTHrP) on the development of cartilaginous bone, we sought to determine whether PTH has any effects on the changes induced by CMA in skeletal growth centers. The interaction between PTH and IGF-I in growth centers during neutral or acidic conditions were studied specifically. An in vitro organ culture system using the murine mandibular condyle was employed as a model for endochondral active growth center. Condyles from six-day-old mice were cultured in BGJb medium of either neutral pH (pH approximately 7.4) or acidic pH (pH approximately 7.15) in the presence or absence of 10-10 mol/L [1-34] PTH. After 24, 48, 72 and 96 hours of culture, the condyles were washed, fixed in formaldehyde, and processed for paraffin embedding. Histologic markers of the growth center were assessed. In addition, the protein level and mRNA expression for various markers of cartilage differentiation were evaluated by immunohistochemistry and in situ hybridization, respectively. The abundance and expression levels of IGF-I and IGF-I receptor (IGF-I-R) were assessed also. Following incubation for 72 hours in acidic conditions, there was a marked attenuation of the chondroblastic zone, suggesting a defect in the process of cellular differentiation. Acidosis also down-regulated endochondral differentiation markers (cartilage specific proteoglycans, collagen type II). This was accompanied by a reduction in the expression of IGF-1, IGF-1 receptor and PTH receptors. PTH (10-10 mol/L) added to acidic cultures prevented the adverse effects of CMA on endochondral differentiation and increased the overall condylar growth, when compared to acidic conditions without PTH. PTH also up-regulated its own receptor in control as well as during acidic conditions, and increased the expression levels of IGF-1 and IGF-1 receptor in the acidotic condyle. Acidosis increased the expression of IGF-I binding protein-4 (IGFBP-4, an inhibitor of IGF-I activity), whereas coincubation with PTH during acidic conditions abrogated the up-regulation of IGFBP-4. Addition of a neutralizing antibody to IGF-I-R during PTH treatment under acidic conditions resulted in the abrogation of the ameliorative effect of PTH on endochondral differentiation. The protein kinase C (PKC) signaling pathway was modulated negatively by CMA. However, PTH activated PKC-alpha under both control and acidic conditions. The phorbol ester, PMA (phorbol 12-myristate 13-acetate), a PKC activator, mimicked the effect of PTH on chondrocyte differentiation. Parathyroid hormone at low concentration stimulates the differentiation and proliferation of cartilage cells and prevents the suppressive effect of acidosis on endochondral bone differentiation and on the IGF-I/IGF-I-R system in skeletal growth centers. Increased local production of IGF-I by PTH, which takes place even during acidotic conditions, mediates, at least in part, the ameliorative effect of PTH. Protein kinase C is probably one of the signaling pathways mediating the salutary effects of PTH on chondrocyte differentiation in growth centers. This study lends further credence to the notion that under certain conditions, PTH or PTHrP can exert anabolic effects in the skeleton. These findings may be of clinical-therapeutic significance in children and patients with CMA.