Chronic acidosis-induced growth retardation is mediated by proton-induced expression of Gs protein.
The etiology of skeletal growth retardation accompanying metabolic acidosis is not clear. Using ex vivo models for endochondral ossification, we showed that the cAMP/PKA pathway, probably triggered by proton sensitive G-protein-coupled receptors, is responsible for impaired skeletal growth in acidosis.


BACKGROUND
Chronic metabolic acidosis (CMA) is very often accompanied by skeletal growth retardation. We have previously shown in an ex vivo model of endochondral ossification that murine mandibular condyles subjected to acidic conditions exhibit growth retardation accompanied by a decline of insulin-like growth factor-I (IGF-I) and its receptors. PTH-induced ameliorative effects on the CMA-induced growth retardation of the mandibular condyle are partially mediated by protein kinase C (PKC). In this study we explored the mechanisms underlying the acidosis-induced growth retardation; in particular, the involvement of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) cellular pathway in the process.


METHODS
Mandibular condyles from neonatal mice or mandibular condyle derived chondrocytes (MCDCs) were incubated for 3 days under either control or acidic conditions or in the presence of cAMP-regulating factors (cAMPrf) such as forskolin, iso-butyl methyl xanthine (IBMX), or 8-Br cAMP. The effects on proliferation and differentiation of the cultures as well as on phosphorylation of cAMP responsive element binding protein (CREB) and increased expression of the alpha subunit, Gs were determined. The intracellular pH was detected using the acridine orange assay.


RESULTS
Our results show that, under acidic conditions, PKA levels were increased. H89 abolished the adverse effects of acidosis on condylar development and restored IGF-I and IGF-I receptors (IGF-IR) levels. The inhibitory effects of acidosis on proliferation and differentiation of cartilaginous cells were mimicked by cAMPrf. We have also shown that acidosis stimulates activation of Gs trimeric protein and CREB phosphorylation. GDPbetaS--a Gs antagonist--abolished the acidosis-induced condylar growth arrest. Using an acridine orange assay, we showed that the intracellular environment is not acidified under acidic conditions.


CONCLUSIONS
Our results indicate that the adverse effects of acidosis on skeletal growth centers are mediated at least in part by the cAMP/PKA cellular pathway. We speculate that high proton concentrations exerted by acidosis conditions stimulate proton sensitive G-protein-coupled receptors, which are mediated by the cellular cAMP/PKA pathway and induce skeletal growth retardation.
Goldberg
Ruth
R
Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
Reshef-Bankai
Ella
E
Coleman
Raymond
R
Green
Jacob
J
Maor
Gila
G
D000138:Acidosis / Q000503:physiopathology*; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002455:Cell Division; D002908:Chronic Disease; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006130:Growth Disorders / Q000378:metabolism* / Q000503:physiopathology; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D008334:Mandible / Q000254:growth & development; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011522:Protons; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology
D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D011522:Protons; D013449:Sulfonamides; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019205:GTP-Binding Protein alpha Subunits, Gs; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide