Acidosis and Protein Kinase: A Novel Mechanism of Growth Failure

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Chronic acidosis is known to cause growth failure by an effect on the bone end-organ, but the exact mechanism has remained elusive. Goldberg and colleagues have recreated growth retardation of endochondrial ossification centers ex vivo by culturing murine mandibular condyles in medium with 2.4 mM HCl, to lower the pH to 7.1 – 7.15. In previous studies, this group found that acidosis led to decreased expression of both insulin-like growth factor (IGF)-I and its receptor (IGF-IR) as well as markers of differentiation like type II collagen and cartilage proteoglycans. They also found that the acidotic growth inhibition could be prevented by local application of low concentrations (10-10 M) of parathyroid hormone (PTH).

PTH works through 2 main signaling pathways: Gq protein/protein kinase C (PKC) and Gs protein/adenylate cyclase/protein kinase A (PKA) pathway. Goldberg and colleagues previously showed that acidosis represses PKC expression, an effect partially inhibited by PTH. However, the PKC agonist PMA succeeded in protecting condyles against acidotic differentiation arrest (increased expression of type II collagen and proteoglycans) but not the acidotic suppression of IGF-I and IGF-IR expression.1 Therefore, the researchers sought to examine the possible role of the PKA pathway in acidosis-induced growth retardation.

In contrast to the reduction in PKC levels, PKAα protein levels were increased by acidosis; both levels were normalized by adding PTH to the acidotic cultures. A specific PKA inhibitor, H89, prevented the acidosis-induced reductions in expression of IGF-I, IGF-IR, and aggrecan (the core protein of cartilage-specific proteoglycans in chondrocytes). Using the converse approach, the cAMP regulating factors 8Br-cAMP (a cAMP analog), iso-butyl methyl xanthine ([IBMX] a phosphodiesterase inhibitor), and forskolin (an adenylate cyclase analog), all reproduced the morphologic changes seen in acidotic growth plates: decreased condylar length with loss of the chondroblast population, leaving the mature hypertrophic cell layer adjacent to the chondroprogenitor zone which is itself wider due to differentiation arrest. Chondrocyte proliferation was also reduced by acidosis, IBMX, and forskolin, as evidenced by decreased expression of proliferating cell nuclear antigen (PCNA), a cell cycle marker. Acidosis and IBMX also decreased expression of IGF-IR in the chondroblasts and chondrocytes. Using mandibular condyle-derived primary cultures of chondrocyte (MCDC) cells, the temporal cascade of endochondrial ossification was reproduced. When grown in acidotic conditions for one week, MCDC cells showed less proliferation and developed fewer cartilaginous nodules. The possibility of toxic effects of acidosis acidifying the intracellular cytoplasm was neatly ruled out by comparing the fluorescence pattern of a pH-dependent fluorescent dye, acridine orange; intracellular pH looked normal despite acidotic culture conditions, but reflected intracellular acidosis with the addition of nigericin (an ionophore that equalizes intracellular and extracellular proton concentrations). Involvement of the entire PKA pathway by acidosis was demonstrated by the increased ratio of phosphorylated-(activated) to total cAMP-responsive element binding protein ([CREB] the transcription factor which is a major PKA substrate) at one end, and the increased expression of Gsα protein at the other. Despite acidosis, the Gs inhibitor GDPβS allowed normal condyle development.

Thus, the authors developed a model of growth plate chondrocytes whereby acidosis induces Gsα protein, which in turn activates adenlyate cyclase and hence PKA, leading to phosphorylated CREB and altered gene expression. Both genes of differentiation and IGF-IR are ultimately down-regulated. PTH inhibits the acidotic growth retardation through both its PKA and PKC signaling pathways. The authors speculated that the activation of Gs protein by acidosis was mediated through proton-sensing receptors rather than ligand binding.

Goldberg R, Reshef-Bankai E, Coleman R, Green J, Maor G. Chronic acidosis-induced growth retardation is mediated by proton-induced expression of Gs protein. J Bone Miner Res. 2006; 21:703–713.

Figure 1. Growth plate and process of growth.

Figure 1. Growth plate and process of growth.

Reprinted with permission from: Grimberg A, De Leon D. Disorders of Growth. In Moshang T, Ed., Requisites in Pediatrics - Pediatric Endocrinology, Elsevier, Inc., Philadelphia, 2005; 127–167. Copyright © Elsevier. 2005. All rights reserved.

Figure 2. Effects of 8Br-cAMP, IBMX, and forskolin (cAMPrf) on the development of the mandibular condyle.

Figure 2. Effects of 8Br-cAMP, IBMX, and forskolin (cAMPrf) on the development of the mandibular condyle. Condyles derived from 6-day-old ICR mice were cultured for 72 h under normal, acidic condition (2.4 mM HCl) or treated with the following cAMP-inducing factors (cAMPrf): 0.05 mM 8BrcAMP (a cAMP analog), 0.05 mM IBMX (a phosphodiesterase inhibitor), or 1 µM forskolin (anadenylyl cyclase analog). Chondroblastic cell layer (CB) is absent in the acidosis and cAMPrf cultures, leaving the hypertrophic cells (HC) adjacent to the chondroperichondrium zone (CP), which is larger than that of the control.

Reprinted with permission from: Goldberg R, et al. J Bone Miner Res. 2006; 21:703–713. Copyright © American Society for Bone and Mineral Research 2006. All rights reserved.

Editor’s Comment

I am always delighted when the underlying mechanisms of long-standing clinical observations are finally elucidated. To remind our readers, the anatomy of the growth plate and the growth process are depicted in the schematic2 of Figure 1. The chondroblasts absent in the condyle cultures with acidosis or cAMP regulating factors in the current paper (Figure 2) correspond to the proliferative zone in Figure 1. This zone is regulated primarily by IGF-I and estradiol. Instead, the hypertrophic zone was seen adjacent to chondroprogenitors (Figure 2) in an expanded reserve zone (Figure 1) that failed to fully differentiate.

Although showing a role of PKA in acidosis-induced growth retardation in the growth plate is certainly novel, this is not the first paper to demonstrate regulation of the IGF axis by PKA. cAMP/PKA induced IGF-I expression in primary rat osteoblasts3 and cultured embryonic mouse mandibular mesenchymal cells,4 IGF binding protein (IGFBP)-1 in hepatocytes,5 and IGFBP-3, -4 and -5 in periosteal and osteoblast bone cell cultures.6 PKA inhibitors interfered with the induction of IGF-I and IGFBP-3 by growth hormone in porcine ovarian granulosa cells,7 IGFBP-3 by FSH also in porcine ovarian granulosa cells,8 and IGFBP-4 by platelet-derived growth factor-BB in fetal rat lung fibroblasts.9

Adda Grimberg , MD

References - (linked to Pubmed Links)

  1. Green J, Goldberg R, Maor G. PTH ameliorates acidosis-induced adverse effects in skeletal growth centers: The PTH-IGF-I axis. Kidney Int. 2003;63:487–500.
  2. Grimberg A, De Leon D. Disorders of Growth. In Moshang T, Ed., Requisites in Pediatrics - Pediatric Endocrinology, Elsevier, Inc., Philadelphia, 2005; 127–167.
  3. Thomas MJ , Umayahara Y , Shu H , Centrella M , Rotwein P , McCarthy TL . Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts. J Biol Chem. 1996;271:21835–21841.
  4. Lambert HW, Weiss ER, Lauder JM. Activation of 5-HT receptors that stimulate the adenylyl cyclase pathway positively regulates IGF-I in cultured craniofacial mesenchymal cells.. Dev. Neurosci. 2001;23:70–77.
  5. Suwanichkul A, DePaolis LA, Lee PD, Powell DR. Identification of a promoter element which participates in cAMP-stimulated expression of human insulin-like growth factor-binding protein-1. J Biol Chem. 1993;268:9730–9736.
  6. Chen Y, Shu H, Ji C, et al. Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone. J Cell Biochem. 1998;71:351–362.
  7. Sirotkin AV, Makarevich AV. Growth hormone can regulate functions of porcine ovarian granulosa cells through the cAMP/protein kinase A system. Anim Reprod Sci. 2002;70:111–126.
  8. Ongeri EM, Verderame MF, Hammond JM. Follicle-stimulating hormone induction of ovarian insulin-like growth factor-binding protein-3 transcription requires a TATA box-binding protein and the protein kinase A and phosphatidylinositol-3 kinase pathways. Mol Endocrinol. 2005;19:1837–1848.
  9. Price WA. PDGF-BB regulates IGF-mediated IGFBP-4 proteolysis in fetal lung fibroblasts. Exp Lung Res. 2001; 27:655–674.

 

 

 


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