Chronic Acidosis-Induced Growth Retardation Is Mediated byProton-Induced Expression of Gs Protein

Ruth Goldberg,1 Ella Reshef-Bankai,1 Raymond Coleman,1 Jacob Green,2 and Gila Maor1

ABSTRACT: The etiology of skeletal growth retardation accompanying metabolic acidosis is not clear. Usingex vivo models for endochondral ossification, we showed that the cAMP/PKA pathway, probably triggered byproton sensitive G-protein–coupled receptors, is responsible for impaired skeletal growth in acidosis.

Introduction: 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 condylessubjected to acidic conditions exhibit growth retardation accompanied by a decline of insulin-like growthfactor-I (IGF-I) and its receptors. PTH-induced ameliorative effects on the CMA-induced growth retardationof the mandibular condyle are partially mediated by protein kinase C (PKC). In this study we explored themechanisms underlying the acidosis-induced growth retardation; in particular, the involvement of the cyclicadenosine monophosphate/protein kinase A (cAMP/PKA) cellular pathway in the process.Materials and Methods: Mandibular condyles from neonatal mice or mandibular condyle derived chondro-cytes (MCDCs) were incubated for 3 days under either control or acidic conditions or in the presence ofcAMP-regulating factors (cAMPrf) such as forskolin, iso-butyl methyl xanthine (IBMX), or 8-Br cAMP. Theeffects on proliferation and differentiation of the cultures as well as on phosphorylation of cAMP responsiveelement binding protein (CREB) and increased expression of the � subunit, Gs were determined. Theintracellular pH was detected using the acridine orange assay.Results: Our results show that, under acidic conditions, PKA levels were increased. H89 abolished the adverseeffects of acidosis on condylar development and restored IGF-I and IGF-I receptors (IGF-IR) levels. Theinhibitory effects of acidosis on proliferation and differentiation of cartilaginous cells were mimicked bycAMPrf. We have also shown that acidosis stimulates activation of Gs trimeric protein and CREB phosphor-ylation. GDP�S—a Gs antagonist—abolished the acidosis-induced condylar growth arrest. Using an acridineorange 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 mediatedat least in part by the cAMP/PKA cellular pathway. We speculate that high proton concentrations exerted byacidosis conditions stimulate proton sensitive G-protein–coupled receptors, which are mediated by the cellularcAMP/PKA pathway and induce skeletal growth retardation.J Bone Miner Res 2006;21:703–713. Published online on February 20, 2006; doi: 10.1359/JBMR.060210

Key words: acidosis, skeletal growth, protein kinase A, forskolin, iso-butyl methyl xanthine, 8Br-cAMP,proton-mediated receptors

INTRODUCTION

METABOLIC ACIDOSIS IS a common consequence of ad-vanced chronic renal failure (CRF) caused by glo-

merulopathies, vascular and tubulointerstitial disorders,and chronic renal tubular disorders.(1,2) Chronic metabolicacidosis (CMA) specifically affects skeletal tissues. CMAdecreases albumin synthesis, induces negative nitrogen bal-ance, and stimulates the catabolism of muscle and bone inexperimental animals and humans. Chronic metabolic aci-dosis exerts profound adverse effects on bone metabolism

in both adult and juvenile skeletal systems. CMA in adultsis involved in the pathogenesis of renal osteodystrophy andosteomalacia(1)as well as in postmenopausal osteoporo-sis.(3) CMA in children suffering from uremic acidosis orrenal tubular acidosis causes growth retardation.(4–7)

Whereas the molecular basis of the CMA-induced adverseeffects in the adult bone is well understood, acidosis effectson bone growth are far less understood. Endochondral os-sification in growth plates proceeds through several con-secutive steps regulated by hormonal (local and systemic)and metabolic factors. These factors generate close cou-pling between proliferation and differentiation, which is aprerequisite for normal linear growth.(8,9) After the activeThe authors state that they have no conflicts of interest.

1Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel;2Department of Nephrology, Rambam Medical Center, Haifa, Israel.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 21, Number 5, 2006Published online on February 20, 2006; doi: 10.1359/JBMR.060210© 2006 American Society for Bone and Mineral Research

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JO505320 703 713 May

proliferation phase, chondroblast cells differentiate intochondrocytes secreting type II collagen and aggrecan. Ma-ture chondrocytes become hypertrophic acquiring an al-tered program of matrix synthesis including type X collagenand calcification.(10–12) This cascade of events is highlyregulated by systemic and local factors mainly by thegrowth hormone/ insulin-like growth factor I (GH/IGF-I)axis.(13–15)

CMA-induced growth retardation has been attributed tomalnutrition-anorexia, low protein-energy intake, and in-creased levels of cortisol and PTH,(16–20) but has beenmainly implicated with interference in the GH/IGF-Iaxis.(6,21–25) Moreover, it has been shown that high concen-tration of IGF-I binding proteins (IGF-IBP), both systemi-cally and locally further contribute to the low active IGF-Ilevels.(26–29) However, the effects of CMA on skeletalgrowth modulating hormones are not conclusive. Bircan etal.(4) claimed that the growth hormone (GH) secretion testin short stature CRF children is not significantly differentfrom age-matched prepubertal healthy short children. It hasalso been shown that growth retardation in CMA childrenis implicated with either secondary hyperparathyroidism(16)

or hypoparathyroidism.(30) Consequently, therapeutic ap-proaches for skeletal growth retardation in CMA childrenbased mainly on GH supplementation(24,31–33) are not verysuccessful.(34) Resistance to GH and IGF-I therapy havealso been shown in acidotic rats. Ordonez et al.(23) reportedthat administration of IGF-I to acidotic rats does not accel-erate their growth “suggesting that a peripheral mechanism,at the level of target tissues, is responsible for the resistanceto the growth-promoting actions of GH and IGF-I.” Usingorgan culture of murine-derived mandibular condylesgrown under acidotic conditions as an ex vivo experimentalmodel of a CMA skeletal growth center,(20) we have shownthat the local resistance to the GH/IGF-I endocrine axis isthe one that counts. Subjecting the condyles to acidic con-ditions results in marked inhibitory effects on the expres-sion of differentiation markers such as type II collagen andcartilage proteoglycans, whereas proliferation seems to beless affected. CMA also exerted a state of resistance to theGH/ IGF-I axis.

We also showed that local application of low concentra-tions (10−10 M) of PTH prevent the CMA-induced harmfuleffects in skeletal growth centers.(26) Thus, PTH reversesthe inhibitory effects of acidosis on the expression of type IIcollagen and cartilage proteoglycans as well as on IGF-Iand its receptor. PTH exerts its biological effects on cellsthrough two signal transduction pathways: Gs protein acti-vates the adenylate cyclase and the downstream proteinkinase A (PKA) cascade and Gq protein activates proteinkinase C (PKC).(35–37) We have also shown that acidosismodulates negatively the expression of PKC and that PTHpartially reverses PKC levels under acidotic conditions.(26)

However, PMA, a PKC agonist, could not entirely abrogatethe CMA-induced adverse effects on the mandibular con-dyle. PMA protected the condyle against the differentiationarrest induced by acidosis reflected in increased expressionof type II collagen and cartilage proteolycans. However,preliminary studies have shown that PMA did not reverse

the acidosis-induced inhibition of IGF-I and IGF-I recep-tors, indicating that PKC by itself is not sufficient to explainthe changes noted in CMA-induced skeletal growth retar-dation.

The aim of this study was to further elucidate the mecha-nisms that mediate the adverse acidotic effects on skeletalgrowth. We found that unlike PKC, PKA levels are mark-edly increased in acidotic cartilage and are normalized byPTH. The PKA antagonist, H89, abolished the acidotic-induced inhibitory effects on skeletal growth, indicatingthat the elevated PKA levels probably play a role in theetiology of the acidotic-induced harmful effects on skeletalgrowth. The acidotic effects could also be mimicked byPKA-associated pathway analogs such as forskolin, whichstimulates adenylate cyclase by a receptor-independentmechanism,(38) and cAMP-regulating factors (cAMPrf)such as 8Br-cAMP and iso-butyl methyl xanthine (IBMX),a phosphodiesterase inhibitor.(39–41) We further showedthat acidosis triggers the entire Gs-mediated cAMP/PKApathway, leading eventually to the unfavorable effects onthe condylar growth. We speculate that these effects aremediated by Gs-coupled proton-sensing receptors.

MATERIALS AND METHODS

Organ culture

Organ culture of mandibular condyles was performed aspreviously described.(20,42–44) Briefly, mandibular condylesderived from 6-day-old ICR mice (Institute of Cancer Re-search, New York, NY, USA) were aseptically dissected,and cultured in the presence of BGJb medium (Fitton Jack-son Modification, Beth Haemek, Israel) supplemented with2% FCS, 100 �g/ml ascorbic acid, and antibiotics. Explantswere incubated for 3 days in the presence or absence of oneof the following: 10−10 M rat PTH(1-34) (cat. No. P1803;Sigma, St Louis, MO, USA), 0.05 mM 8Br cAMP (a cAMPanalog), 0.05 mM IBMX (a phosphodiesterase inhibitor),and 1 �M forskolin (an adenylate cyclase agonist) or 50 �Mguanosine 5-o-(2-thiodiphosphate) (GDP-�-S, G7637;Sigma) (antagonist of Gs), under acidic or normal condi-tions. Acidosis (pH 7.1–7.15) was achieved by addition of2.4 mM HCl to the medium. At the end of the incubationperiod, the explants were thoroughly washed with coldHank’s buffer, fixed with neutral buffered formalin, androutinely processed for paraffin embedding.

Alcian blue + H&E staining

Paraffin sections (6 �m) were deparaffinized in xylene,hydrated in graduated ethanols, and pretreated with 3%acetic acid for 3 minutes. Sections were stained with 1%alcian blue at pH 2.5 for 30 minutes, thoroughly rinsed withtap water, and counterstained with H&E.

Tissue culture

Mandibular condyle derived primary cultures (MCDC)were prepared as previously described.(45) Briefly, man-dibular condyles derived from 3-day-old ICR mice wereharvested aseptically, freed of any soft tissue, cut at the

GOLDBERG ET AL.704

mineralization front of the condyle, washed in cold Hank’sbuffer, and subjected to graduated collagenase digestion(type II collagenase; Sigma). Homogenous cell populationwas cultured in DMEM supplemented with 100 �g/mlascorbic acid, 10 mM �-glycerophosphate, 1 mM calciumchloride, 1 mM sodium pyruvate, 10% FCS, and antibiotics.Cells were plated at a concentration of 5 × 105 cells/ml in35-mm 6-well culture dishes under normal or acidic (2.4mM HCl) conditions, and the medium was changed every48 h.

MTT analysis

Viability of the cells was assessed by MTT assay. Cellswere incubated for 2 h in the presence of 1.25 mg/ml [3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT, Sigma)—a substrate of mitochondrial dehydroge-nase active only in intact cells . The blue formazan productwas solubilized with 0.4N isopropanol/HCl and read at 570nm.

Monitoring intracellular pH

The intracellular pH of the acidotic culture was moni-tored using the acridine orange assay. Acridine orange is asensitive optical probe used to follow pH gradient over cellmembranes.(46) The monoamine acridine orange accumu-lates on the inner side of the membranes and fluorescencesdifferently under different pH conditions. When excitedwith 488 nm, it emits at 530 nm (green) at neutral pH andat 630 nm (orange) under acidic (2.4 mM HCl) pH.(47,48)

MCDC culture (5 × 105 cells/ml) was incubated under con-trol or acidotic conditions. For positive controls, cultureswere treated with 1 mM nigericin in the presence of 130mM KCl for 2 minutes and in the presence or absence of 2.4mM HCl. Nigericin is a K+/H+ ionophore pump used tobalance the intra- and extracellular pH levels. Acridine or-ange (6 �M) was added for 2 minutes, and the fluorescencewas visualized by inverted fluorescence microscopy (Axio-vert 135; Zeiss).

Immunohistochemistry

Immunohistochemistry (IHC) was performed as de-scribed elsewhere.(20,42–44) Deparaffinized paraffin sectionswere reacted for 2 h at room temperature with antibodiesagainst one of the following antigens: IGF-I receptor(anti-� subunit, cat. no. sc-712; Santa Cruz Biotechnology,Santa Cruz, CA, USA), type II collagen (cat. no. MAB8887; Chemicon International), type X collagen (cat. no.M0879; Dako), or proliferating cell nuclear antigen (PCNA;cat. no. SS 852-P1; NeoMarkers). This was followed by in-cubation with an appropriate biotinylated second antibody,streptavidin-peroxidase conjugate, and S-(2-aminoethyl)-L-cysteine (AEC) as a substrate (Histostain-SP kit; ZymedLaboratory, San Francisco, CA, USA); counterstaining wasdone with hematoxylin. For collagen analysis, sections werepredigested 10 minutes with 1 mg/ml pepsin (in Tris buffer,pH 2.0).

Densitometry

Quantification of IHC (and in situ hybridization [ISH])staining was performed using densitometry software

(Image-Pro Plus 5.0; Media Cybernetics, Silver Springs,MD, USA). Each value represents a mean of at least 18measurements performed on three sections from each of sixreplicates (condyles) cultured in three different experi-ments. Values are expressed as integrated optical density(IOD).

SDS-PAGE and Western blotting

Lysate proteins prepared from pooled(10–12) 3-day-oldcultured mandibles by homogenizing with 20 × 20-s strokesin ice cold radioimmunoprecipitation assay (RIPA) buffersupplemented with protease inhibitors, phenyl methylsulphonyl fluoride (PMSF) 10 �g/ml, aprotonin 5 �g/ml,trypsin inhibitor 5 �g/ml, benzamidine 10 �g/ml, were sepa-rated in a reducing SDS-PAGE and electrotransferred tonitrocellulose membrane. Blots were incubated with anti-bodies against the following antigens: PKA� (cat. no. sc-903; Santa Cruz Biotechnology), IGF-I receptor (seeabove), PCNA (see above), CREB1 (cat no. sc-186; SantaCruz Biotechnology), p-CREB1 (cat no. sc-7978; SantaCruz Biotechnology), Gs� (cat no. 371731; Calbiochem),and �-actin (cat no. MAB 1501; Chemicon International) asa loading control. Detection was performed using relevanthorseradish peroxidase (HRP)–conjugated second anti-body (Zymed Laboratory), and revealed by chemilumines-cence reagent (New England Nuclear). For each antigentested, immunoblotting analysis was repeated with threedifferent lysate proteins (each derived from pooled con-dyles as described above).

ISH

ISH was performed as previously described.(20,42–44) De-paraffinized sections on precleaned poly-L-lysine–coatedslides were treated with 3% H2O2 in methanol to neutralizeendogenous peroxidase. After permeabilization with 12.5�g/ml proteinase K for 15 minutes (stopped with 2 mg/mlglycine) and acetylation in 0.5% acetic anhydride in 0.1 MTris at pH 8.0, sections were postfixed with 4% paraform-aldehyde/PBS. Prehybridization for 10 minutes in 2× SSCwas followed by 1 h in hybridization buffer: 50% formam-ide, 0.5 mg/ml salmon sperm DNA, 4× SSC, 1× Denhardt,200 U/ml heparin, 5% dextran sulfate, and 0.01% SDS.Hybridization was done overnight (18 h) at 42°C and maxi-mal humidity with a 5 �g/ml digoxygenin-labeled antisenseRNA probe. Digoxygenin-labeled sense RNA probes wererun as negative controls (data not shown). At the end of theincubation period, slides were rinsed in SSC under increas-ingly stringent conditions and then with 0.1 M Tris and 0.15M NaCl at pH 7.5. Hybrids were detected using anti-digoxygenin antibodies conjugated with peroxidase (Boe-hringer Mannheim) and AEC as a substrate; counterstain-ing was done with hematoxylin.

Densitometry quantification of the ISH-positive stainingwas performed as described above.

Digoxygenin-labeled antisense RNA probes for ISH

We used a probe for the core protein of the cartilagespecific proteoglycan (aggrecan) cloned in pBluescript SK+

amp+ (922 bp). After linearization, digoxygenin-labeled an-

CHRONIC ACIDOSIS-INDUCED GROWTH RETARDATION 705

tisense RNA was transcribed using the (Sp6/T7) Dig-RNAlabeling kit (Boehringer Mannheim), according to themanufacturer’s instructions.

Morphometry

Morphometric analyses were performed using Axiovert135 (Zeiss) and Image-Pro-Plus software (Media Cybernet-ics). Significance was determined using Student’s t-testanalysis.

RESULTS

Acidosis-induced increase of PKA� and decrease inPKC levels

We have previously shown that PKC levels in acidoticcondyles are significantly low and that the PTH-inducedrecovery effects on CMA-treated cartilage are accompa-nied by PKC upregulation. To elucidate further the signal-ing pathways involved in the acidotic-induced impaired de-velopment of the mandibular condyles that might beimplicated in the CMA-induced growth retardation, we ex-amined the levels of PKA� in acidotic condyles in compari-son with those of PKC. Densitometry of immunoblottinganalysis (Table 1) showed that, unlike its inhibitory effectson PKC (33% compared with control), acidosis increasesPKA� levels by 42% over control values. These rather un-expected results indicate that acidosis-induced high levelsof PKA� may be a causative factor for skeletal growthretardation. PTH normalized both PKC and PKA levels ofthe acidotic cultures. PKC levels were increased by 35% (p< 0.02) and PKA levels were decreased by 23% (p < 0.05)over the levels of the acidotic cultures alone. Both PKC andPKA levels of PTH-treated acidic cultures were not signifi-cantly different from those of the relative controls.

H89 (PKA inhibitor) abolishes the acidosis-inducednegative effects on the development of themandibular condyle

The role played by PKA� in mediating the acidosis-induced harmful effects on condylar growth was studiedusing H89, a specific PKA inhibitor (Fig. 1). Immunohisto-chemical analysis of 3-day-old cultures revealed that levelsof IGF-I and IGF-I receptor (IGF-IR), both expressed inchondroprogenitor and chondrocytes, were markedly de-creased under acidic conditions. ISH analysis revealed thataggrecan expression mainly expressed in chondrocytes wasmarkedly reduced in the acidotic cells. The intensity ofstaining was assessed using densitometric analysis. The rela-tive staining intensity, shown in Fig. 1, indicates that acido-sis reduces the levels of IGF-I, IGF-IR, and CSPG by 36%,

22%, and 20%, respectively, compared with controls andthat H89 reverses the amounts of these parameters to thecontrol levels.

TABLE 1. EFFECTS OF PTH ON THE ACIDOSIS-INDUCED CHANGES

IN THE LEVELS OF PKA AND PKC (DENSITOMETRY OF

IMMUNOBLOTTING ANALYSIS NORMALIZED TO ACTIN)

Control Acidosis Acidosis + PTHPKC/actin 300 ± 15 201 ± 18 315 ± 22PKA�/actin 295 ± 22 420 ± 25 320 ± 15

FIG. 1. Effects of H89 on the acidosis-induced adverse effects onthe development of mandibular condyles. Mandibular condylesderived from 6-day-old ICR mice were cultured for 72 h undercontrol, acidic conditions, or acidic conditions + 10−6 M H89. De-paraffinized sections were used for analysis of functional param-eters. Levels of (A) IGF-I and (B) IGF-IR expressed in bothchondroprogenitors (CP) and chondrocytes (CH) were monitoredimmunohistochemically using relevant antibodies. (C) Expressionof the core protein of cartilage-specific proteoglycans (aggrecan)in the chondrocytes (CH) was detected by in situ hybridizationusing digoxygenin-labeled CSPG antisense probe. Densitometryof the positive staining shows that acidosis reduces the levels ofIGF-I, IGF-IR, and CSPG by 36%, 22%, and 20%, respectively,compared with the control (*p < 0.05), and H89 reversed thelevels of these parameters to the control values. Results representthree different experiments, three to four measurements in eachexperiment.

GOLDBERG ET AL.706

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High cAMP concentrations mimic the adverseeffects of CMA on the condylar development

To further elucidate the role of PKA� in mediating aci-dosis-induced growth inhibition, we studied the effects ofvarious cAMP regulating factors (cAMPrf) on the endo-chondral ossification process. After preliminary dose–response studies, we examined the effects of 3-day treat-ment of the following: cAMPrf, 0.05 mM 8BrcAMP (acAMP analog), 0.05 mM IBMX (a phosphodiesterase in-hibitor), and 1 �M forskolin (an adenylate cyclase agonist),on the development of the mandibular condyle. Results ofH&E + Alcian blue staining, seen in Fig. 2A, show that all

three factors induce morphological changes similar to thoseinduced by acidic conditions. The chondroblast populationcompletely disappears, leaving the mature hypertrophic cellpopulation adjacent to the chondroprogenitor zone. Thelatter seems more extensive than that of the untreated con-trols, indicating clear differentiation arrest.

Morphometric analysis of the overall length of the con-dyles seen in Fig. 2B shows that acidosis causes a decreaseof 15%. 8Br cAMP, IBMX, and forskolin decreased con-dylar length by 13.3%, 23.5%, and 18.2%, respectively.These results clearly indicate that cAMPrf mimics acidosisin condylar growth inhibition. Measurements of the chon-

FIG. 2. Effects of 8Br-cAMP, IBMX, and forskolin (cAMPrf) on the development of the mandibular condyle. Condyles derived from6-day-old ICR mice were cultured for 72 h under normal, acidic condition (2.4 mM HCl) or treated with the following cAMP-inducingfactors (cAMPrf): 0.05 mM 8BrcAMP (a cAMP analog), 0.05 mM IBMX (a phosphodiesterase inhibitor), or 1 �M forskolin (anadenylyl cyclase analog). (A) Cultures were fixed and processed for paraffin embedding. Paraffin sections stained with Alcian blue +H&E show that the 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. (B) Morphometric analysis of the overalllength of the condyles shows that acidosis causes a decrease of 15%. 8Br cAMP, IBMX, and forskolin decrease condylar length by13.3%, 23.5%, and 18.2%, respectively. These results clearly indicate that cAMPrf mimics acidosis in condylar growth inhibition. (C)Measurements of the chondroprogenitor zone width reveal significant increase by both acidosis and cAMPrf. Acidosis increases thechondroprogenitor zone by 24%; 8Br cAMP, IBMX, and forskolin increase the chondroprogenitor zone by 44%, 88%, and 92%,respectively. Each result represents an average ± SD of 12–18 measurements performed on six to nine condyles cultured in threedifferent experiments. *p < 0.05; **p < 0.02; ***p < 0.01. Measurements were performed on histological sections obtained from fourdifferent experiments. (D) Type II collagen synthesis was determined using IHC. Paraffin sections were subjected to predigestion with1 mg/ml pepsin (in Tris buffer, pH 2.0) for 10 minutes followed by routine processing for immunohistochemistry using mouse anti-typeII collagen (MAB 8887; Chemicon International) antibody. Acidosis and markedly diminish type II collagen staining compared withthe control, whereas the effects of 8Br cAMP and IBMX are milder.

CHRONIC ACIDOSIS-INDUCED GROWTH RETARDATION 707

Fig 2 live 4/C

droprogenitor zone width (Fig. 2C) reveal significant in-creases in both acidosis and cAMPrf. Acidosis increasedthe chondroprogenitor zone by 24%; 8Br cAMP, IBMX,and forskolin increased the chondroprogenitor zone by44%, 88%, and 92%, respectively. Each result representsan average of 12–18 measurements performed on six tonine condyles cultured in three different experiments.These results may be caused by either increased prolifera-tion or arrest of chondroblastic differentiation causing ac-cumulation of precursor cells in the chondroprogenitorzone.

The impact of acidosis and cAMPrf on chondrocytic dif-ferentiation was determined by assaying its effects on thelevels of type II collagen (Fig. 2D). It seems that both aci-dosis and forskolin markedly reduced the expression oftype II collagen. The smaller inhibitory effects of IBMXand 8 Br-cAMP may reflect a state of “leakage” of phos-phodiesterase molecules in the case of IBMX and limitedagonist effects of 8 Br-cAMP on the expression of type IIcollagen.

Effects of cAMPrf on cell proliferation

The reciprocal effects on the overall condylar size andthe width of the progenitor zones probably represent theuncoupling between proliferation and differentiation. Totest this, we studied the effects of acidosis and cAMP regu-lating factors on the expression of PCNA, a DNA polymer-ase co-factor that correlates with cell cycle. Results of im-munoblotting analysis depicted in Fig. 3 show that acidosis,IBMX, and forskolin significantly reduced the PCNA+ re-action by 14%, 18.5%, and 20%, respectively.

Effects of cAMPrf on IGF-I receptors

The major local regulator of chondrogenesis is IGF-I.Mediated by its specific receptors (IGF-IR), IGF-I regu-lates both proliferation and differentiation. To study thechanges in the IGF-I axis, the effects of cAMPrf on thelevels of IGF-I receptors were determined (Fig. 4). Consis-tent with earlier observations,(25) the inhibitory effects ofacidosis are mainly apparent in the chondroprogenitor andearly chondrocyte zones. Immunoblotting of the IGF-I re-ceptor normalized to actin showed that acidosis and IBMXreduced the levels of IGF-IR by 16% and 22%, respectively(p < 0.05). These rather slight, but significant, decreases inIGF-IR indicate that the inhibitory effects of cAMPrf on thecondylar growth are partially mediated by IGF-I receptors.

Effect of acidosis on the intracellular pH ofMCDC cells

To further elucidate the cellular mechanisms that regu-late the cAMPrf-induced inhibitory effects on chondrogen-esis, we studied the effects of acidosis on the developmentof cultures of primary chondrocytes in a culture model ofMCDCs, which follows a strict temporal cascade of endo-chondral ossification processes. Preliminary results haveshown that growing MCDC cultures under acidic condi-tions for 7 days results in a significant reduction in theMTT+ cells and in the number and extent of cartilaginousnodules. It seems that, similar to the effects observed in theorgan culture model, acidosis interferes with both prolifera-tion and differentiation of the MCDC culture.

This primary chondrocytic culture model allows determi-nation of the effects of extracellular acidosis on intercellular

FIG. 3. PCNA expression in acidosis and cAMPrf-treatedmandibular condyles. The effects of acidosis, forskolin, and IBMXon the proliferation activity of the condyles were assessed by fol-lowing the expression of PCNA. Immunoblotting (IB) of lysateproteins obtained from condyles cultured under parallel con-ditions. Densitometry results of PCNA-IB normalized to actinrevealed 14%, 18.5%, and 20% reduction in the expressionof PCNA under acidosis, forskolin, and IBMX, respectively.Each value represents an average of three different experiments.*p < 0.05; **p < 0.02.

FIG. 4. Levels of IGF-I receptors (IGF-IR). Mandibular con-dyles derived from 6-day-old mice were cultured for 3 days underacidotic conditions or treated with IBMX as described in Fig. 3.Levels of IGF-IR were detected in paraffin sections by (A–C)immunohistochemistry or (D) in lysate proteins by immunoblot-ting. Both acidosis and IBMX decreased the expression of IGF-IRin the chondroblasts and chondrocytes (B and C vs. A). (D) Im-munoblotting analysis of IGF-IR normalized to actin revealed areduction of 16% and 22% in the acidosis and IBMX-treatedcondyles compared with the control. Each value represents anaverage of three different experiments. *p < 0.05.

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pH. High intracellular proton concentrations may causenonspecific/toxic effects. To refute the possibility that aci-dosis-induced inhibitory effects on cartilage developmentare caused by cytoplasm acidification of the cells causingtoxic effects, we monitored intracellular pH under acidicconditions. For this purpose, we used acridine orange(AO), a monoamine-pH sensitive fluorescent dye, whichunder acidic conditions shows orange fluorescence and atneutral pH emits green fluorescence. Acidotic and controlMCDC cultures were treated with AO as previously de-scribed and were observed immediately for the emittingfluorescence. For positive controls, cultures were pre-treated with nigericin, which equalizes the intracellular andextracellular proton concentrations. Figure 5 clearly showsthat, without nigericin, the intracellular pH under acidosis issimilar to that of control conditions. Nigericin added to theacidotic culture increased the intracellular pH as reflectedin the orange fluorescence of the cells. These results verifythat the acidosis-induced inhibitory effects on cartilage tis-sues are specific/nontoxic effects.

PKA and p-CREB levels in the acidoticMCDC culture

To determine the cellular pathways that mediate the aci-dosis-induced effects on MCDC development, we studiedthe levels of the catalytic subunit of PKA� under acidosisand IBMX-treated cultures (Fig. 6A). Immunoblottinganalysis of 3-day-old cultures show that acidosis and IBMXincrease PKA� levels normalized to actin by 36% (p < 0.05)

and 61% (0.02), respectively, compared with the untreatedcultures. These results are consistent with those obtained inthe mandibular condyle organ cultures (see Table 1). More-over, this effect can be mimicked by IBMX, emphasizingthe major role of cAMP in the process.

To elucidate further the involvement of the cAMP/PKApathway in acidosis-mediated chondrocytic growth inhibi-tion, we studied the effects of acidosis on the phosphoryla-tion of transcription factor CREB, which serves as one ofthe major PKA substrates. The levels of pCREB normal-ized to the levels of total CREB were determined by im-munoblotting. Results (Fig. 6B) show that both acidosis andIBMX increase the levels of phosphorylated CREB (28%,p < 0.05 and 80%, p < 0.01, respectively), indicating thatacidosis stimulates the whole cAMP/PKA cascade in medi-ating the acidosis effects on chondrocytes.

FIG. 5. Effects of acidosis on intracellular pH of MCDC cells.MCDC cultures were incubated for 24 h under (A and C) controlor (B and D) acidic (2.4 mM HCl) conditions in the (C and D)presence or (A and B) absence of 1 mM of the ionophore L-ni-gericin in the presence of 130 mM KCl for the last 2 minutes ofincubation. Acridine orange (6 �M) was added for 2 minutes, andthe fluorescence (excited with 488 nm) was visualized by invertedfluorescence microscopy (Axiovert 135; Carl Zeiss). (A–C) Neu-tral intracellular pH yields green fluorescence; (D) acidic intra-cellular pH yields orange fluorescence.

FIG. 6. Activation of PKA� and CREB in the MCDC cultures.MCDC cultures were incubated for 3 days under control or acidic(2.4 mM HCl) conditions or in the presence of 0.05 mM IBMX.Lysate proteins served for immunoblotting analysis of PKA� us-ing (A) a specific anti-activated form of PKA� antibody (sc-903;Santa-Cruz) and (B) pCREB (sc-7978; Santa-Cruz). (A) Acidosisand IBMX increase levels of PKA-normalized to actin by 36%,and 61%, respectively, and (B) the levels of pCREB normalizedto CREB by 28% and 80%, respectively, In both cases, each valuerepresents an average of three different experiments. *p < 0.05;**p < 0.02. Experiments were repeated four times.

CHRONIC ACIDOSIS-INDUCED GROWTH RETARDATION 709

Fig 5 live 4/C

Effects of acidosis on the activity of Gs protein

Extracellular signals that stimulate the cAMP/PKA path-way activate the Gs� subunit, which dissociates from the�-� subunits and stimulates adenylyl cyclase to form cAMP,which activates PKA. We next studied the effects of acido-sis on the levels of the Gs� subunit using immunoblottingand anti-Gs� antibody. Results obtained from three differ-ent experiments (Fig. 7) showed that acidosis increases thelevels of Gs� by 45% (p < 0.005) over the untreated con-trols, indicating that high extracellular proton concentra-tions may stimulate the expression of the Gs� protein.

Blocking of Gs by GDPßS

To further verify the role played by the membrane Gsprotein in mediating the acidosis effects on skeletal growth,we examined the effects of GDP�S (a Gs blocker) on aci-dotic mandibular condyles. Results shown in Table 2 indicatethat mandibular condyles cultured for 6 days under acidoticconditions in the presence of GDP�S develop normally.Morphometric studies indicate that the overall length of theacidotic condyle is 20% smaller (p < 0.02) than that of thecontrol. Addition of GDP�S reverses the acidosis-inducedgrowth inhibition, resulting in normal growth velocity. Eachresult represents an average of 18 measurements performedon six condyles cultured in three different experiments.

DISCUSSION

Our previous studies have shown that PTH preventsCMA-induced adverse effects in skeletal growth centers.

Using a mandibular condyle organ culture system as an exvivo model for endochondral ossification, we have shownthat low PTH doses induces upregulation of IGF-I, IGF-Ireceptors, and their beneficial effects in growth centers.(26)

PTH exerts its biological effects through two major cellularpathways: the cAMP-dependent PKA and the diacylglyc-erol (DAG)-stimulated PKC pathways. These pathways aremediated by two isoforms of the � subunit: Gs and Gq.(49)

Our previous studies have also shown that PTH reversesthe CMA-induced decrease of PKC levels and therebyameliorates the acidosis-induced arrest of chondrocytic dif-ferentiation. However, the CMA-induced attenuation ofthe IGF-I system and its effects on proliferation were notentirely reversed by PMA (a PKC activator), indicating thata mechanism other than partial PKC inhibition is respon-sible for acidosis-induced skeletal growth retardation.

In this study, we further explored the cellular mecha-nisms involved in CMA-mediated skeletal growth retarda-tion, focusing on the involvement of activated Gs in theprocess. Surprisingly, we found that, unlike its effect onPKC, acidosis markedly increases the levels of PKA.Hence, the notion that high levels of PKA are involved inthe acidosis-induced skeletal growth retardation was chal-lenged in this study. H89 (a PKA antagonist) completelynegated the acidosis-mediated condylar growth arrest. ThecAMP/PKA pathway can be triggered by increasing intra-cellular cAMP levels. This was achieved by using the fol-lowing cAMP regulatory factors (cAMPrfs) normally usedin skeletal tissues: forskolin (an adenylyl cyclase activator),8Br-cAMP (a cAMP analog), and IBMX (a cAMP phos-phodiesterase antagonist). Increasing cAMP concentrationsin the condylar chondrocytes interferes with normal differ-entiation of the cartilage cells, resulting in a total disappear-ance of the chondroblast population, a marked increase ofthe chondroprogenitor zone, and a significant decrease inthe overall condylar length, thus resembling the effects ofCMA on condylar development. These effects are accom-panied by a decrease in the expression of PCNA and typeII collagen.

The cAMP/PKA pathway has been implicated with vari-ous catabolic activities in growing cartilage accompanied byaccelerated maturation and enhanced osteogenesis. It hasbeen shown that IL-17–induced catabolism of cartilage inrheumatoid arthritis is mediated by PKA. Inhibition of ret-inoic acid receptor (RAR)-mediated PKA is crucial for theexpression of SOX9, which serves as a major regulator ofearly chondrogenesis. PKA also interferes in transglutamin-ase-mediated mineralization of young chick-derived chon-drocytes and is involved in PTH-related protein (PTHrP)-induced suppression of Cbfa1, a major regulator of earlychondrogenesis.(50–52) In addition, Li et al.(53) have shownthat prostaglandin E2 (PGE2) mediated inhibition of en-dochondral ossification through activated PKA. It has also

FIG. 7. Levels of Gs� in MCD cells. MCDC cultures incubatedunder control or acidic (2.4 mM HCl) conditions serve for immu-noblotting detection of the activated form of Gs� using specificanti-activated Gs� antibody (371731; Calbiochem). Acidosis in-creases levels of Gs� normalized to actin by 45%, (**p < 0.005).Experiments were repeated three times.

TABLE 2. EFFECTS OF GDP�S ON THE DEVELOPMENT OF

MANDIBULAR CONDYLES (MORPHOMETRY ANALYSIS, �m)

Control Acidosis Acidosis + GDP�S605 ± 15 484 ± 15 600 ± 25

GOLDBERG ET AL.710

been shown the PTHrP-induced downregulation of Runx2(Cbfa1) and its inhibitory effect on Indian hedgehog(IHH)-mediated differentiation are PKA dependent.(52)

Other reports, however, have shown that, in some cases,PKA is implicated in anabolic effects on chondrogenic ac-tivities. Phosphorylation of SOX9 by PKA in mouse em-bryo–derived chondrocytes enhances its transcriptional andDNA-binding activity, and by doing so, SOX9 regulates theproliferation of cartilage precursor cells.(54) Using a modelof mouse limb mesenchymal cells, Weston et al.(51) haveshown that activation of RAR signaling, which attenuatesdifferentiation, can be rescued by activation of p38 MAPKor PKA, indicating that PKA generates beneficial effects onchondrogenesis. The apparent discrepancy in the above re-ports may stem from the different developmental stages ofthe experimental systems used in these studies. Usually el-evated PKA levels in postnatal skeletal tissues are impli-cated with catabolic effects, whereas PKA anabolic effectsare observed only in embryonic/mesenchymal cells. TheMCDCs, as in most of the postnatal endochondral ossifica-tion models, are markedly inhibited by high PKA levelsinduced by acidosis.

These PKA inhibitory effects are not caused by nonspe-cific acidification of the cytoplasm of chondrocytes, as isapparent from the green fluorescence of the AO-stainedacidotic cells. This fluorescent dye accumulates on the innerside of the cell membrane and fluorescences differently un-der different pH conditions.(55)

In view of these results, we studied the involvement ofthe cAMP/PKA signaling cascade in the acidosis-inducedinhibition of chondrogenesis. Similarly to their effects onthe mandibular organ culture, acidosis and IBMX also in-creased PKA levels in MCDC culture. Both these agentsalso increased phosphorylation of CREB-binding protein.The sustained phosphorylation state of CREB shown after3 days might be caused by downregulation of ICER, aninducible cAMP early repressor, or NIPP1, a nuclear in-hibitor of protein phosphatase-1.(56,57) Although thesecAMP-related regulatory factors have not been implicatedin cartilage development, their modulation by acidosis willbe studied in the future. However, because PKA and PKCcellular cascades accompany cartilage development, theirmodulation by cartilage regulatory factors may be sustainedfor days.(51,58) CREB has been shown to be a critical me-diator in modulation of development of chondrocytes.(59) Ithas also been shown that okadaic acid induces chondrogen-esis in chicken limb buds, PTHrP inhibits chondrocytematuration, PGE2 inhibits differentiation of chondrocytes,and Smad3 induces chondrogenesis through CREB phos-phorylation.(52,53,60) In cAMP responsive cells, heterotri-meric G proteins transduce signals from various cell surfacereceptors to adenylyl cyclases, which generate cAMP. Ourresults show that acidosis significantly increases the expres-sion of the Gs� subunit of the trimeric G protein. More-over, blocking the action of Gs� by the antagonistGDP�S(61–63) totally abolishes the acidosis-induced growtharrest and restores the typical developmental gradient inthe acidotic condyle.

These results indicate that acidosis conditions modulatethe Gs-mediated cAMP/PKA pathway cascade in chondro-

cytes of growth centers, interfering with normal differentia-tion processes and leading to growth arrest. Gs protein ac-tivity under acidic conditions is probably not ligandmediated, but is more likely activated through proton-sensing receptors. Proton sensitive G-protein–coupled re-ceptors (GPCRs) have been shown to exist in a variety ofpH responsive cells. T cell death-associated gene 8 is aGPCR mainly expressed in lymphoid organs and cancertissues; activation of this proton sensitive GPCR upregu-lates the cAMP/PKA pathway.(64–66) Ludwig et al.(67) haveshown that skeletal tissues that participate in pH homeo-stasis as a buffering organ contain some binding sites, whichare proton sensitive and are coupled to the Gs-mediatedcAMP cellular pathway, thus emphasizing the role of pro-ton-sensitive receptors in pH regulating tissues.

Overall results reported here and the studies referencedlead us to believe that cartilage cells, belonging to the skel-etal system, also contribute to pH buffering and conse-quently possess proton-sensitive Gs coupling receptors.These receptors respond to low pH by activating the Gs-mediated cAMP/PKA pathway. In chronic metabolic aci-dosis, prolonged low pH levels markedly elevate the cellu-lar PKA cascade, leading to hindrance of normalchondrocytic differentiation and eventually to skeletalgrowth arrest.

REFERENCES

1. Bushinsky DA 1995 The contribution of acidosis to renal os-teodystrophy. Kidney Int 47:1816–1832.

2. Carlisle EJ, Donnelly SM, Halperin ML 1991 Renal tubularacidosis (RTA): Recognize the ammonium defect and pHorgetthe urine pH. Pediatr Nephrol 5:242–248.

3. Frassetto L, Morris RC Jr, Sebastian A 1997 Potassium bicar-bonate reduces urinary nitrogen excretion in postmenopausalwomen. J Clin Endocrinol Metab 82:254–259.

4. Bircan Z, Kervancioglu M, Soran M, Yildirim I 1998 Growthhormone stimulation tests in chronic renal failure with meta-bolic acidosis. Acta Paediatr Jpn 40:70–72.

5. Kuizon BD, Salusky IB 1999 Growth retardation in childrenwith chronic renal failure. J Bone Miner Res 14:1680–1690.

6. Brungger M, Hulter HN, Krapf R 1997 Effect of chronic meta-bolic acidosis on the growth hormone/IGF-1 endocrine axis:New cause of growth hormone insensitivity in humans. KidneyInt 51:216–221.

7. Groothoff JW, Offringa M, Van Eck-Smit BL, Gruppen MP,Van De Kar NJ, Wolff ED, Lilien MR, Davin JC, HeymansHS, Dekker FW 2003 Severe bone disease and low bone min-eral density after juvenile renal failure. Kidney Int 63:266–275.

8. Stevens DA, Williams GR 1999 Hormone regulation of chon-drocyte differentiation and endochondral bone formation. MolCell Endocrinol 151:195–204.

9. Ohlsson C, Isgaard J, Tornell J, Nilsson A, Isaksson OG, Lin-dahl A 1993 Endocrine regulation of longitudinal bone growth.Acta Paediatr Suppl 82(Suppl 391):33–41.

10. Yamasaki A, Itabashi M, Sakai Y, Ito H, Ishiwari Y, Nagat-suka H, Nagai N 2001 Expression of type I, type II, and type Xcollagen genes during altered endochondral ossification on thefemoral epiphysis of osteosclerotic (oc/oc) mice. Calcif TissueInt 68:53–60.

11. Poole AR, Kojima T, Yasuda T, Mwale F, Kobayashi M,Laverty S 2001 Composition and structure of articular carti-lage: A template for tissue repair. Clin Orthop 391:S26–S33.

12. Poole AR, Matsui Y, Hinek A, Lee ER 1989 Cartilage mac-romolecules and the calcification of cartilage matrix. Anat Rec224:167–179.

13. Maor G, Hochberg Z, Silbermann M 1989 Growth hormone

CHRONIC ACIDOSIS-INDUCED GROWTH RETARDATION 711

stimulates the growth of mouse neonatal condylar cartilage invitro. Acta Endocrinol (Copenh) 120:526–532.

14. Takigawa M, Shirai E, Enomoto M, Kinoshita A, Pan HO,Suzuki F, Yugari Y 1989 Establishment from mouse growthcartilage of clonal cell lines with responsiveness to parathyroidhormone, alkaline phosphatase activity, and ability to producean endothelial cell growth inhibitor. Calcif Tissue Int 45:305–313.

15. Siebler T, Robson H, Shalet SM, Williams GR 2001 Glucocor-ticoids, thyroid hormone and growth hormone interactions:Implications for the growth plate. Horm Res 56(Suppl 1):7–12.

16. Sanchez CP 2003 Secondary hyperparathyroidism in childrenwith chronic renal failure: Pathogenesis and treatment. Paedi-atr Drugs 5:763–776.

17. Wiederkehr MR, Kalogiros J, Krapf R 2004 Correction ofmetabolic acidosis improves thyroid and growth hormone axesin haemodialysis patients. Nephrol Dial Transplant 19:1190–1197.

18. Guarnieri G, Toigo G, Fiotti N, Ciocchi B, Situlin R, GiansanteC, Vasile A, Carraro M, Faccini L, Biolo G 1997 Mechanismsof malnutrition in uremia. Kidney Int Suppl 62:S41–S44.

19. Cupisti A, D’Alessandro C, Morelli E, Rizza GM, Galetta F,Franzoni F, Barsotti G 2004 Nutritional status and dietary ma-nipulation in predialysis chronic renal failure patients. J RenNutr 14:127–133.

20. Green J, Maor G 2000 Effect of metabolic acidosis on thegrowth hormone/IGF-I endocrine axis in skeletal growth cen-ters. Kidney Int 57:2258–2267.

21. Salusky IB, Kuizon BG, Juppner H 2004 Special aspects ofrenal osteodystrophy in children. Semin Nephrol 24:69–77.

22. Al-Owain M, Freehauf C, Bernstein L, Kappy M, Thomas J2004 Growth hormone deficiency associated with methylmalo-nic acidemia. J Pediatr Endocrinol Metab 17:239–243.

23. Ordonez FA, Santos F, Martinez V, Garcia E, Fernandez P,Rodriguez J, Fernandez M, Alvarez J, Ferrando S 2000 Resis-tance to growth hormone and insulin-like growth factor-I inacidotic rats. Pediatr Nephrol 14:720–725.

24. Jehle S, Hulter HN, Krapf R 2000 On the mechanism of growthhormone-induced stimulation of renal acidification in humans:Effect of dietary NaCl. Clin Sci (Lond) 99:47–56.

25. Challa A, Krieg RJ Jr, Thabet MA, Veldhuis JD, Chan JC 1993Metabolic acidosis inhibits growth hormone secretion in rats:Mechanism of growth retardation. Am J Physiol 265:E547–E553.

26. Green J, Goldberg R, Maor G 2003 PTH ameliorates acidosis-induced adverse effects in skeletal growth centers: The PTH-IGF-I axis. Kidney Int 63:487–500.

27. McCarty MF 2004 Chronic activation of AMP-activated kinaseas a strategy for slowing aging. Med Hypotheses 63:334–339.

28. Jandziszak K, Suarez C, Wasserman E, Clark R, Baker B, LiuF, Hintz R, Saenger P, Brion LP 1998 Disturbances of growthhormone-insulin-like growth factor axis and response togrowth hormone in acidosis. Am J Physiol 275:R120–R128.

29. Levitt Katz LE, Satin-Smith MS, Collett-Solberg P, ThorntonPS, Baker L, Stanley CA, Cohen P 1997 Insulin-like growthfactor binding protein-1 levels in the diagnosis of hypoglycemiacaused by hyperinsulinism. J Pediatr 131:193–199.

30. Mahlbacher K, Sicuro A, Gerber H, Hulter HN, Krapf R 1999Growth hormone corrects acidosis-induced renal nitrogenwasting and renal phosphate depletion and attenuates renalmagnesium wasting in humans. Metabolism 48:763–770.

31. Sanchez CP, Salusky IB, Kuizon BD, Abdella P, Juppner H,Goodman WG 1998 Growth of long bones in renal failure:Roles of hyperparathyroidism, growth hormone and calcitriol.Kidney Int 54:1879–1887.

32. Sanchez CP, Kuizon BD, Goodman WG, Gales B, EttengerRB, Boechat MI, Wang Y, Elashoff R, Salusky IB 2002Growth hormone and the skeleton in pediatric renal allograftrecipients. Pediatr Nephrol 17:322–328.

33. Greenbaum LA, Del Rio M, Bamgbola F, Kaskel F 2004 Ra-tionale for growth hormone therapy in children with chronickidney disease. Adv Chronic Kidney Dis 11:377–386.

34. Kotzmann H, Yilmaz N, Lercher P, Riedl M, Schmidt A,

Schuster E, Kreuzer S, Geyer G, Frisch H, Horl WH, Mayer G,Luger A 2001 Differential effects of growth hormone therapyin malnourished hemodialysis patients. Kidney Int 60:1578–1585.

35. Schwartz Z, Semba S, Graves D, Dean DD, Sylvia VL, BoyanBD 1997 Rapid and long-term effects of PTH(1-34) on growthplate chondrocytes are mediated through two different path-ways in a cell-maturation- dependent manner. Bone 21:249–259.

36. Goltzman D 1999 Interactions of PTH and PTHrP with thePTH/PTHrP receptor and with downstream signaling path-ways: Exceptions that provide the rules. J Bone Miner Res14:173–177.

37. Amizuka N, Henderson JE, White JH, Karaplis AC, GoltzmanD, Sasaki T, Ozawa H 2000 Recent studies on the biologicalaction of parathyroid hormone (PTH)- related peptide(PTHrP) and PTH/PTHrP receptor in cartilage and bone. His-tol Histopathol 15:957–970.

38. Insel PA, Ostrom RS 2003 Forskolin as a tool for examiningadenylyl cyclase expression, regulation, and G protein signal-ing. Cell Mol Neurobiol 23:305–314.

39. Ogata Y, Nakao S, Kim RH, Li JJ, Furuyama S, Sugiya H,Sodek J 2000 Parathyroid hormone regulation of bone sia-loprotein (BSP) gene transcription is mediated through a pi-tuitary-specific transcription factor-1 (Pit-1) motif in the ratBSP gene promoter. Matrix Biol 19:395–407.

40. Kiroytcheva M, Cheval L, Carranza ML, Martin PY, Favre H,Doucet A, Feraille E 1999 Effect of cAMP on the activity andthe phosphorylation of Na+,K(+)-ATPase in rat thick ascend-ing limb of Henle. Kidney Int 55:1819–1831.

41. Dobashi K, Asayama K, Shirahata A 2003 Differential effectsof cyclic AMP on induction of nitric oxide synthase in 3T3-L1cells and brown adipocytes. Free Radic Biol Med 35:94–101.

42. Maor G, Segev Y, Phillip M 1999 Testosterone stimulates in-sulin-like growth factor-I and insulin-like growth factor-I-receptor gene expression in the mandibular condyle–a modelof endochondral ossification. Endocrinology 140:1901–1910.

43. Maor G, Rochwerger M, Segev Y, Phillip M 2002 Leptin actsas a growth factor on the chondrocytes of skeletal growth cen-ters. J Bone Miner Res 17:1034–1043.

44. Maor G, Karnieli E 1999 The insulin-sensitive glucose trans-porter (GLUT4) is involved in early bone growth in controland diabetic mice, but is regulated through the insulin-likegrowth factor I receptor. Endocrinology 140:1841–1851.

45. Reiter I, Tzukerman M, Maor G 2002 Spontaneous differenti-ating primary chondrocytic tissue culture: A model for endo-chondral ossification. Bone 31:333–339.

46. Palmgren MG 1991 Acridine orange as a probe for measuringpH gradients across membranes: Mechanism and limitations.Anal Biochem 192:316–321.

47. Altan N, Chen Y, Schindler M, Simon SM 1999 Tamoxifeninhibits acidification in cells independent of the estrogen re-ceptor. Proc Natl Acad Sci USA 96:4432–4437.

48. Hunter SJ, Schraer H, Gay CV 1988 Characterization of iso-lated and cultured chick osteoclasts: The effects of acetazol-amide, calcitonin, and parathyroid hormone on acid produc-tion. J Bone Miner Res 3:297–303.

49. Swarthout JT, D’Alonzo RC, Selvamurugan N, Partridge NC2002 Parathyroid hormone-dependent signaling pathwaysregulating genes in bone cells. Gene 282:1–17.

50. Martel-Pelletier J, Mineau F, Jovanovic D, Di Battista JA,Pelletier JP 1999 Mitogen-activated protein kinase and nuclearfactor kappaB together regulate interleukin-17-induced nitricoxide production in human osteoarthritic chondrocytes: Pos-sible role of transactivating factor mitogen-activated proteinkinase-activated proten kinase (MAPKAPK). ArthritisRheum 42:2399–2409.

51. Weston AD, Chandraratna RA, Torchia J, Underhill TM 2002Requirement for RAR-mediated gene repression in skeletalprogenitor differentiation. J Cell Biol 158:39–51.

52. Li TF, Dong Y, Ionescu AM, Rosier RN, Zuscik MJ, SchwarzEM, O’Keefe RJ, Drissi H 2004 Parathyroid hormone-related

GOLDBERG ET AL.712

peptide (PTHrP) inhibits Runx2 expression through the PKAsignaling pathway. Exp Cell Res 299:128–136.

53. Li TF, Zuscik MJ, Ionescu AM, Zhang X, Rosier RN, SchwarzEM, Drissi H, O’Keefe RJ 2004 PGE2 inhibits chondrocytedifferentiation through PKA and PKC signaling. Exp Cell Res300:159–169.

54. Huang W, Zhou X, Lefebvre V, de Crombrugghe B 2000 Phos-phorylation of SOX9 by cyclic AMP-dependent protein kinaseA enhances SOX9’s ability to transactivate a Col2a1 chondro-cyte-specific enhancer. Mol Cell Biol 20:4149–4158.

55. Konttinen YT, Mandelin J, Li TF, Salo J, Lassus J, LiljestromM, Hukkanen M, Takagi M, Virtanen I, Santavirta S 2002Acidic cysteine endoproteinase cathepsin K in the degenera-tion of the superficial articular hyaline cartilage in osteoarthri-tis. Arthritis Rheum 46:953–960.

56. De Cesare D, Sassone-Corsi P 2000 Transcriptional regulationby cyclic AMP-responsive factors. Prog Nucleic Acid Res MolBiol 64:343–369.

57. De Cesare D, Fimia GM, Sassone-Corsi P 1999 Signalingroutes to CREM and CREB: Plasticity in transcriptional acti-vation. Trends Biochem Sci 24:281–285.

58. Nurminskaya M, Magee C, Faverman L, Linsenmayer TF 2003Chondrocyte-derived transglutaminase promotes maturationof preosteoblasts in periosteal bone. Dev Biol 263:139–152.

59. Long F, Schipani E, Asahara H, Kronenberg H, Montminy M2001 The CREB family of activators is required for endochon-dral bone development. Development 128:541–550.

60. Furumatsu T, Tsuda M, Taniguchi N, Tajima Y, Asahara H2005 Smad3 induces chondrogenesis through the activation ofSOX9 via CREB-binding protein/p300 recruitment. J BiolChem 280:8343–8350.

61. Ho RJ, Shi QH, Ruiz J 1986 Conditional inhibition of forsko-lin-activated adenylate cyclase by guanosine diphosphate andits analog. Arch Biochem Biophys 251:148–155.

62. Oberdisse E, Lapetina EG 1987 GDP beta S enhances theactivation of phospholipase C caused by thrombin in human

platelets: Evidence for involvement of an inhibitory GTP-binding protein. Biochem Biophys Res Commun 144:1188–1196.

63. Bastepe M, Weinstein LS, Ogata N, Kawaguchi H, Juppner H,Kronenberg HM, Chung UI 2004 Stimulatory G protein di-rectly regulates hypertrophic differentiation of growth platecartilage in vivo. Proc Natl Acad Sci USA 101:14794–14799.

64. Wang JQ, Kon J, Mogi C, Tobo M, Damirin A, Sato K, Ko-machi M, Malchinkhuu E, Murata N, Kimura T, Kuwabara A,Wakamatsu K, Koizumi H, Uede T, Tsujimoto G, Kurose H,Sato T, Harada A, Misawa N, Tomura H, Okajima F 2004TDAG8 is a proton-sensing and psychosine-sensitive G-pro-tein-coupled receptor. J Biol Chem 279:45626–45633.

65. Radu CG, Nijagal A, McLaughlin J, Wang L, Witte ON 2005Differential proton sensitivity of related G protein-coupled re-ceptors T cell death-associated gene 8 and G2A expressed inimmune cells. Proc Natl Acad Sci USA 102:1632–1637.

66. Ishii S, Kihara Y, Shimizu T 2005 Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor. J Biol Chem 280:9083–9087.

67. Ludwig MG, Vanek M, Guerini D, Gasser JA, Jones CE,Junker U, Hofstetter H, Wolf RM, Seuwen K 2003 Proton-sensing G-protein-coupled receptors. Nature 425:93–98.

Address reprint requests to:Gila Maor, DSc

Department of Anatomy and Cell BiologyRappaport Faculty of Medicine

Technion-Israel Institute of TechnologyPO Box 9649

Haifa 31096, IsraelE-mail: gimaor@tx.technion.ac.il

Received in original form May 18, 2005; revised form January 30,2006; accepted February 16, 2006.

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