Control of parathyroid cell growth by calcimimetics

Transcription

1 Nephrol Dial Transplant (2003) 18 [Suppl 3]: iii13 iii17 DOI: /ndt/gfg1004 Control of parathyroid cell growth by calcimimetics Michihito Wada and Nobuo Nagano Pharmaceutical Development Laboratories, Kirin Brewery Co. Ltd, Takasaki-shi, Gunma, Japan Abstract Parathyroid cell hyperplasia is commonly observed in patients with chronic renal insufficiency and largely accounts for refractory secondary hyperparathyroidism. Calcimimetics are newly synthesized compounds that activate a calcium receptor on the parathyroid cell and can suppress parathyroid hormone secretion. The calcimimetic compound AMG 073 has been examined in clinical trials, and the data obtained so far demonstrate that the compound can lower the circulating levels of parathyroid hormone and calcium phosphorus product in patients with secondary hyperparathyroidism. Furthermore, experimental evidence indicates that calcimimetics have the potential to inhibit parathyroid cell proliferation and block the progression of parathyroid hyperplasia. These beneficial effects, especially the potential to control parathyroid cell proliferation, would place calcimimetics among the essential therapeutic agents for treating secondary hyperparathyroidism. Keywords: calcimimetics; calcium receptor; chronic renal insufficiency; hyperparathyroidism; parathyroid hormone; parathyroid hyperplasia Introduction Management of parathyroid hyperplasia in chronic renal insufficiency (CRI) is still a challenge [1 3]. Despite intensive use of calcitriol therapy, secondary hyperparathyroidism often progresses over the long term from diffuse (hyperplastic) to nodular (adenomatous) forms that are refractory to conventional medical therapy [4,5]. Small organic molecules named calcimimetics that can enhance the sensitivity of the calcium receptor (CaR) to extracellular Ca 2q (Ca 2q o) on parathyroid cells have been identified [6,7], and their efficacy and safety have been examined in clinical trials [8 11]. Calcimimetics can suppress parathyroid Correspondence and offprint requests to: M. Wada, PhD, Pharmaceutical Development Laboratories, Kirin Brewery Co. Ltd, Takasaki-shi, Gunma , Japan. wadam@kirin.co.jp hormone (PTH) secretion, and these compounds would be expected to improve medical treatment of hyperparathyroidism secondary to CRI [8 11]. Moreover, experimental evidence indicates that calcimimetics have the potential to inhibit parathyroid cell proliferation and block the progression of parathyroid hyperplasia [12 15]. This review summarizes the pharmacological studies evaluating the inhibition of parathyroid cell proliferation by calcimimetics. These data demonstrate that the CaR plays an essential role in regulating parathyroid cell proliferation [16,17]. Also, this review examines the intracellular signalling pathways linking the CaR and cell proliferation. The finding that calcimimetics can suppress parathyroid cell proliferation is important in understanding the pathogenesis of parathyroid hyperplasia and is of significant therapeutic relevance. Calcium receptor and the control of PTH secretion The parathyroid glands play an important role in maintaining the plasma Ca 2q o concentration within a narrow physiological range [18,19]. Brown et al. identified a G-protein-coupled receptor (GPCR) that functions as a key element in the parathyroid Ca 2q o-sensing mechanism, called the calcium-sensing receptor or calcium receptor because it recognizes Ca 2q oas its physiological ligand [20]. The CaR consists of an extracellular domain N-terminal of ;600 amino acids, a central core of ;250 amino acids with seven transmembrane domains, and an intracellular C-terminal tail of ;200 amino acids [19,20]. The CaR is coupled with the G-proteins, G11, Gq and probably one or more isoforms of Gi, which participate in the activation of phosphatidylinositol-specific phospholipase C (PLC) and the inhibition of adenylate cyclase [19,21]. The primary role of the CaR is the continuous control of PTH secretion in response to Ca 2q o concentration on a minute-to-minute basis, but the mechanism underlying this process has not yet been fully elucidated [18,19]. Activation of the CaR by Ca 2q o increases the activity of PLC, thereby producing inositol 1,4,5-trisphosphate and diacylglycerol, which # 2003 European Renal Association European Dialysis and Transplant Association

2 iii14 mobilize intracellular Ca 2q [6,18]. The increases in diacylglycerol and intracellular Ca 2q concentration cause the activation of protein kinase C (PKC) [19]. Kifor et al. reported that cytosolic phospholipase A 2 (cpla 2 ) is phosphorylated by mitogen-activated protein kinase (MAPK), at least in part, in a PKCdependent manner [22]. Thus, the PKC cpla 2 pathway may be involved in the regulation of PTH secretion, because arachidonic acid, produced from phospholipids by the action of cpla 2, and its further metabolites, e.g. hydroxyperoxyeicosatetranoic acids, can suppress PTH secretion [23]. However, the effect of Ca 2q o on PTH secretion is relatively unaffected by inhibitors of PKC, or when PKC activity has been down-regulated, indicating a trivial role for PKC in the control of PTH secretion [6]. Furthermore, little is known about the mechanism of these changes in the process of PTH exocytosis [19]. Calcimimetics suppress PTH secretion and parathyroid cell proliferation Ligands that mimic or potentiate the effects of Ca 2q at the CaR have been termed calcimimetics [6]. Fendiline, a phenylalkylamine derivative, was found to activate the CaR, and the modification of this prototype molecule resulted in the identification of the calcimimetics NPS R-467, NPS R-568, and AMG 073 [7]. These calcimimetics can potentiate the response of the CaR to Ca 2q o and suppress PTH secretion [7,11]. NPS R-568 suppresses PTH secretion in rats with or without secondary hyperparathyroidism [11,24,25] and in patients with primary or secondary hyperparathyroidism [8,9]. Another beneficial effect of calcimimetics is that PTH-related bone diseases such as osteitis fibrosa ameliorate as a long-term outcome of suppressed PTH levels in rats with CRI [26,27] and in ovariectomized rats [28]. However, clinical data indicate that NPS R-568 has limited bioavailability and undergoes metabolic clearance by the polymorphic cytochrome P450 enzyme, CYP2D6 [8,9]. To address these issues, a related but improved compound, AMG 073, was selected for further study [10,29]. The efficacy and safety of AMG 073 have been examined in clinical trials, and the results demonstrate that this calcimimetic can lower both serum PTH concentration and calcium phosphorus product in patients with secondary hyperparathyroidism [10,29]. Growing experimental evidence indicates that calcimimetics can inhibit parathyroid cell growth in vitro and in vivo. In a short-term experiment, treatment with NPS R-568 inhibited parathyroid cell proliferation that had been accelerated in partially nephrectomized rats [12]. This result was confirmed by long-term experiments showing that the development of parathyroid hyperplasia is almost completely blocked in partially nephrectomized rats by treating them with NPS R-568 immediately, or 11 weeks after nephrectomy [13,15]. Apoptotic cell death was not observed in M. Wada and N. Nagano any parathyroid glands of rats treated with NPS R-568 or vehicle [12]. Moreover, it has been shown recently that AMG 073 also inhibits parathyroid cell proliferation and prevents parathyroid hyperplasia in rats with CRI [14]. The effects of both NPS R-568 and AMG 073 occur despite unchanged serum vitamin D and increased phosphorus concentrations (because of lowered PTH), indicating a direct role for the CaR in regulating parathyroid cell proliferation [12 15]. Finally, the direct in vitro antiproliferative effect of NPS R-467 on parathyroid cells has been demonstrated in human parathyroid cells maintained in long-term culture [30]. These findings support the proposal that calcimimetics can inhibit parathyroid cell proliferation and block the development of hyperplasia. Role of calcium in parathyroid cell proliferation In 1997, the antiproliferative effect of NPS R-568 was described [12], although the role of the CaR in regulating parathyroid cell proliferation was still speculative [1,3]. The link between the CaR and parathyroid cell proliferation was suggested because severe neonatal hyperparathyroidism and occasional cases of familial hypocalciuric hypercalcaemia, both of which are caused by inactivating mutations in the CaR, develop parathyroid hyperplasia [16]. Despite this compelling evidence, the role of the CaR remained unclear because the effects of Ca 2q o on parathyroid cell proliferation were debatable [2]. Recent advances, especially those obtained using calcimimetics, may help to reconcile any discrepancy among the results obtained by manipulating Ca 2q o. The secretion of PTH is regulated reciprocally by the concentration of Ca 2q o, and this feedback loop is essential in maintaining systemic Ca 2q homeostasis [18,19]. The Ca 2q o PTH feedback loop is thought to operate on two time scales, i.e. the regulation of PTH secretion and synthesis in the short term, and the control of cell proliferation in the long term [3]. It is reasoned that prolonged demand for PTH creates the compensatory need for a greater number of cells, and hypocalcaemia is the most likely proximate stimulator of parathyroid cell growth, as inferred by Marine in 1914 [31] and subsequently extended by many investigators [3]. This classical view has been questioned [2], because the in vitro effect of Ca 2q o on parathyroid cell proliferation is inconsistent (Table 1). Several studies have shown the inhibitory effect of Ca 2q o on cell proliferation [32 36], but this has not been a consistent finding [37 39]. However, these results may be discounted because dispersed parathyroid cells in the primary culture system lose their response to Ca 2q o, probably by the down-regulation of CaR expression [16,19]. Recently, Roussanne et al. reported that proliferation of cultured human parathyroid cells was stimulated by an increase in Ca 2q o concentration, whereas it was inhibited by the calcimimetic NPS R-467 [30]. In this case, the persistence of functionally

3 Parathyroid cell growth and calcimimetics Table 1. Effect of extracellular Ca 2q on parathyroid cell proliferation in vitro iii15 Effect Species (cell line) Culture system Indices Sources (reference) Suppress Chicken Organ Mitosis Raisz [32] Suppress Rat Organ 3 H incorporation Lee and Roth [33] Suppress Bovine Long term 3 H incorporationucell number Brandi [34] Suppress (PT-r) a 3 H incorporationucell number Sakaguchi [35] Suppress Human First passage 3 H incorporation Liu [36] No effect Bovine Primary Cell number LeBoff [37] No effect Bovine Primary 3 H incorporationucell number Kremer [38] No effect Bovine Primary Cell number Ishimi [39] Stimulate b Human Long term 3 H incorporation Roussanne [30] a Clonal rat parathyroid cell line that secretes PTHrP instead of PTH. b NPS R-467 suppressed cell proliferation. active CaR expression was observed. Given that Ca 2q o has multiple CaR-independent effects, this result has relevance for the hypothesis that only the CaR-specific pathway primarily mediates the antiproliferative effect of Ca 2q o on parathyroid cells. Another cellular response that complicates understanding of the mechanism of Ca 2q o in parathyroid cell proliferation is that hypertrophy, not hyperplasia, mainly accounts for the increase in parathyroid cell mass in animals with normal renal function fed either a calcium-deficient or phosphate-rich diet [1,2]. The alternative view holds that hypocalcaemia does not stimulate parathyroid cell proliferation and does not account for the parathyroid hyperplasia observed in CRI [2]. However, this view is at odds with the findings that the number of proliferating cell nuclear antigen (PCNA)- or Ki67-positive cells significantly increases in animals with hypocalcaemia [40 42]. An important finding is that the calcimimetic NPS R-568 inhibits parathyroid cell proliferation in rats with CRI when given as either an intermittent (once-daily bolus) or a persistent regimen (twice-daily bolus or continuous infusion) [12 15], whereas parathyroid cell hypertrophy is only suppressed when NPS R-568 is administered persistently [12,13,15]. This provides circumstantial evidence suggesting that the duration (and possibly magnitude) of CaR activation determines whether the control of cell size or of proliferation will be the outcome, and the former possibly needs longer activation of the CaR. Depending on the severity of hypocalcaemia, therefore, it is likely that parathyroid hypertrophy will predominate, but this does not necessarily contradict the role of CaR in regulating parathyroid cell proliferation. Signalling pathway linking the calcium receptor and cell proliferation The proliferation of several cell types is stimulated by activation of the CaR [2,19]. Among multiple intracellular signalling pathways, extracellular signal-regulated kinases (ERKs) or MAPKs mediate the proliferative effects of the GPCR [43]. Diverse GPCR ligands can activate p42 and p44 MAPK (known as ERK2 and ERK1, respectively) in many cellular systems [19,43]. The first indication of a role for tyrosine kinases in the pathway linking the GPCR to Ras MAPK was the ability of tyrosine kinase inhibitors to reduce the activation of ERK by Gi-coupled GPCR. In this signalling cascade, rapid tyrosine phosphorylation of Shc (Src homologue and collagen) following GPCR stimulation leads to the formation of Shc Grb2 (growth factor receptor-bound 2) complexes [43]. The CaR also uses this tyrosine kinase Ras pathway to activate ERK in some cell types, because it has been found that the loss-of-function mutation of the CaR results in diminished Src activation in CaR-transfected Rat-1 fibroblast cells [44]. In parathyroid cells also, the activation of tyrosine kinases partially contributes to ERK activation through the Gi and Gbc protein pathway of the CaR [22]. However, the CaR uses multiple signalling cascades to activate MAPK where at least PKC and phosphatidylinositol 3-kinase (PI3kinase) play a key role in parathyroid cells [22,45]. The activation of ERK1u2 elicited by the increase in Ca 2q o concentration or the addition of a calcimimetic compound is almost completely blocked by PKC inhibitors in a PTXinsensitive manner, indicating that the Gqu11 PLC PKC cascade plays a major role in bovine [22] and human parathyroid cells [45]. Human adenomatous parathyroid cells, even in a resting state, have constitutively high ERK activity that occurs in a PKCdependent manner [45]. These results indicate that PKC is an important mediator to activate ERK downstream of the CaR. Furthermore, apart from the PKC cascade, PI3kinase, possibly activated by Gbc, also contributes to the activation of ERK in human parathyroid cells [45]. The downstream cascade of PI3kinase is not defined in parathyroid cells, but it is suggested that PI3kinase acts upstream of the Ras Raf pathway in ovarian epithelial cells [46]. All available data thus indicate that activation of the CaR stimulates the activation of ERK through multiple signalling cascades even in parathyroid cells [21,22,45]. Kifor et al. have reported an important role for caveolin that functions as a scaffolding protein and coordinates the CaR and other signalling molecules to activate ERK in parathyroid cells [21].

4 iii16 Mode of action of calcimimetics The growth control of parathyroid cells is a complex process, and whether the regulation by the CaR is direct or secondary to autocrine (or paracrine) mechanisms still remains unclear. Two lines of evidence tend to favour the latter possibility, although this is far from confirmed. First, the observations that activation of the CaR results both in the inhibition of parathyroid cell growth and in the activation of MAPK are apparently paradoxical and rather compatible with the indirect role of the CaR. Nonetheless, it should be noted that the activation of MAPK does not always stimulate cell proliferation but sometimes inhibits it [47,48]. For example, somatostatin receptor subtypes 1 and 2 activate MAPK, but inhibit cell proliferation by up-regulating the cyclin-dependent kinase inhibitor p21 [47,48]. Secondly, it has been hypothesized that a number of candidate molecules act as autocrine or paracrine regulators of parathyroid cell proliferation in responding to Ca 2q o, but their individual roles remain to be examined [2]. These candidate molecules include parathyroid hormone-related protein [49], endothelin [42], acidic fibroblast growth factor [35] and transforming growth factor-a (TGF-a) [50]. For example, in rats fed a calcium-deficient diet, parathyroid cell growth is blocked by an endothelin antagonist, which does not occur in animals fed a normal diet, inferring that endothelin is a mediator of hypocalcaemia-induced parathyroid cell growth [42]. Likewise, the proliferation of parathyroid cells and the up-regulation of TGF-a are both suppressed in rats with CRI fed a calcium-rich diet, suggesting that TGF-a may play a role in the pathogenesis of parathyroid hyperplasia in CRI, and the increased concentration of Ca 2q o may reverse this process [50]. These results indicate that complex autocrine or paracrine mechanisms may underlie the control of parathyroid cell growth by the CaR, particularly in the setting of pathological hyperplasia. Therefore, at present, it is prudent to state that the effect of calcimimetics on parathyroid cell proliferation is direct through activating the CaR, but the full picture of subsequent molecular events remains to be clarified. Conclusion The results obtained so far indicate that the calcimimetics NPS R-467, NPS R-568 and AMG 073 can inhibit parathyroid cell proliferation by activating the CaR, which completely blocks the development of hyperplasia in rats with CRI. Given that parathyroid hyperplasia is difficult to control with conventional therapies, these findings are therapeutically important. There is still much to understand about the molecular mechanisms underlying the control of parathyroid cell growth by the CaR, however. Finally, the data from clinical trials demonstrate that calcimimetics have potential as therapeutic agents for treating the hyperparathyroidism resulting from CRI. Note The calcimimetic compound AMG 073 is also referred to as KRN1493 in Japan, and these are the same molecule (cinacalcet hydrochloride). Acknowledgements. We would like to thank Drs E. F. Nemeth, J. Fox (NPS Pharmaceuticals, Inc.) and M. R. Downing (Amgen Inc.) for discussion throughout the experiments, their valuable comments and encouragement. References M. Wada and N. Nagano 1. Drüeke TB. The pathogenesis of parathyroid gland hyperplasia in chronic renal failure. Kidney Int 1995; 48: Drüeke TB. Cell biology of parathyroid gland hyperplasia in chronic renal failure. J Am Soc Nephrol 2000; 11: Parfitt AM. The hyperparathyroidism of chronic renal failure: a disorder of growth. Kidney Int 1997; 52: Slatopolsky E, Brown A, Dusso A. Pathogenesis of secondary hyperparathyroidism. Kidney Int 1999; 56 [Suppl. 73]: S14 S19 5. Rodriguez M, Canalejo A, Garfia B, Aguilera E, Almaden Y. Pathogenesis of refractory secondary hyperparathyroidism. Kidney Int 2002; 61: S155 S Nemeth EF. Calcium receptors as novel drug targets. In: Bilezikian, JP, Raisz, LG, Rodan, GA, eds. Principles of Bone Biology. Academic Press, San Diego: 2002; Nemeth EF, Steffey ME, Hammerland LG et al. Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA 1998; 95: Goodman WG. Recent development in the management of secondary hyperparathyroidism. Kidney Int 2001; 59: Coburn JW, Maung HM. Calcimimetic agents and the calciumsensing receptor. Curr Opin Nephrol Hypertens 2000; 9: Frazao JM, Martins P, Coburn JW. The calcimimetic agents: perspectives for treatment. Kidney Int 2002; 61 [Suppl. 80]: S149 S Wada M, Nagano N, Nemeth EF. The calcium receptor and calcimimetics. Curr Opin Nephrol Hypertens 1999; 8: Wada M, Furuya Y, Sakiyama J-I et al. The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency: control of parathyroid cell growth via a calcium receptor. J Clin Invest 1997; 100: Wada M, Nagano N, Furuya Y et al. Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. Kidney Int 2000; 57: Wada M, Kawata T, Furuya Y et al. The calcimimetic compound KRN1493 suppresses parathyroid hyperplasia in rats with chronic renal insufficiency [abstract]. J Bone Miner Res 2002; 17 [Suppl 1]: S Chin J, Miller SC, Wada M et al. Activation of the calcium receptor by a calcimimetic compound halts the progression of secondary hyperparathyroidism in uremic rats. J Am Soc Nephrol 2000; 11: Brown EM, Chattopadhyay N, Vassilev PM, Hebert SC. The calcium-sensing receptor (CaR) permits Ca 2q to function as a versatile extracellular first messenger. Recent Prog Horm Res 1998; 53: Silver J, Kilav R, Naveh-Many T. Mechanisms of secondary hyperparathyroidism. Am J Physiol 2002; 283: F367 F Brown EM. Extracellular Ca 2q -sensing, regulation of parathyroid cell function, and role of Ca 2q and other ions as extracellular (first) messengers. Physiol Rev 1991; 71: Brown EM, MacLeod J. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001; 81:

5 Parathyroid cell growth and calcimimetics 20. Brown EM, Gamba G, Riccardi D et al. Cloning and characterization of an extracellular Ca 2q -sensing receptor from bovine parathyroid. Nature 1993; 366: Kifor O, Kifor I, Brown EM. Signal transduction in the parathyroid. Curr Opin Nephrol Hypertens 2002; 11: Kifor O, MacLeod RJ, Diaz R et al. Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaRtransfected HEK293 cells. Am J Physiol 2001; 280: F291 F Bourdeau A, Moutahir M, Souberbielle J-C et al. Effects of lipoxygenase products of arachidonate metabolism on parathyroid hormone secretion. Endocrinology 1994; 135: Fox J, Lowe SH, Conklin RL, Nemeth EF. Calcimimetic compound NPS R-568 decreases plasma PTH in rats with mild and severe renal or dietary secondary hyperparathyroidism. Endocrine 1999; 10: Fox J, Lowe SH, Petty BA, Nemeth EF. A type II calcimimetic compound that acts on parathyroid cell calcium receptor of rats to reduce plasma levels of parathyroid hormone and calcium. J Pharmacol Exp Ther 1999; 290: Wada M, Ishii H, Furuya Y et al. NPS R-568 halts or reverses osteitis fibrosa in uremic rats. Kidney Int 1998; 53: Ishii H, Wada M, Furuya Y et al. Daily intermittent decreases in serum levels of parathyroid hormone have an anabolic-like action on the bones of uremic rats with low-turnover bone and osteomalacia. Bone 2000; 26: Miller MA, Fox J. Daily transient decrease in plasma parathyroid hormone levels induced by the calcimimetic NPS R-568 slows the rate of bone loss but does not increase bone mass in ovariectomized rats. Bone 2000; 27: Goodman WG, Hladik GA, Turner SA et al. The calcimimetic agent AMG073 lowers plasma parathyroid hormone levels in hemodialysis patients with secondary hyperparathyroidism. J Am Soc Nephrol 2002; 13: Roussanne M-C, Lieberherr M, Souberbielle JC et al. Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate. Eur J Clin Invest 2001; 31: Marine D. Parathyroid hypertrophy and hyperplasia in fowls. Proc Soc Exp Biol Med 1914; 11: Raisz LG. Regulation by calcium of parathyroid growth and secretion in vitro. Nature 1963; 197: Lee MJ, Roth SI. Effect of calcium and magnesium on deoxyribonucleic acid synthesis in rat parathyroid glands in vitro. Lab Invest 1975; 33: Brandi ML, Fitzpatrick LA, Coon HG, Aurbach GD. Bovine parathyroid cells: cultures maintained for more than 140 population doublings. Proc Natl Acad Sci USA 1986; 83: Sakaguchi K. Acidic fibroblast growth factor autocrine system as a mediator of calcium-regulated parathyroid cell growth. J Biol Chem 1992; 267: Liu W, Ridefelt P, Akerstrom G, Hellman P. Differentiation of human parathyroid cells in culture. J Endocrinol 2001; 168: iii LeBoff MS, Rennke HG, Brown EM. Abnormal regulation of parathyroid cell secretion and proliferation in primary culture of bovine parathyroid cells. Endocrinology 1983; 113: Kremer R, Bolivar I, Goltzman D, Hendy GN. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 1989; 125: Ishimi Y, Russell J, Sherwood LM. Regulation by calcium and 1,25-(OH) 2 D 3 of cell proliferation and function of bovine parathyroid cells in culture. J Bone Miner Res 1990; 5: Naveh-Many T, Rahamimov R, Livni N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats: the effects of calcium, phosphate, and vitamin D. J Clin Invest 1995; 96: Wang Q, Palnitkar S, Parfitt AM. Parathyroid cell proliferation in the rat: effect of age and phosphate administration and recovery. Endocrinology 1996; 137: Kanesaka Y, Tokunaga H, Iwashita K et al. Endothelin receptor antagonist prevents parathyroid cell proliferation of low calcium diet-induced hyperparathyroidism in rats. Endocrinology 2001; 142: Van Biesen T, Luttrell LM, Hawes BE, Lefkowitz RJ. Mitogenic signaling via G protein-coupled receptors. Endocr Rev 1996; 17: McNeil SE, Hobson SA, Nipper V, Rodland KD. Functional calcium-sensing receptors in rat fibroblasts are required for activation of Src kinase and mitogen-activated protein kinase in response to extracellular calcium. J Biol Chem 1998; 273: Corbetta S, Lania A, Filopanti M et al. Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells. J Clin Endocrinol Metab 2002; 87: Bilderback TR, Lee F, Auersperg N, Rodland KD. Phosphatidylinositol 3-kinase-dependent, MEK-independent proliferation in response to CaR activation. Am J Physiol 2002; 283: C282 C Florio T, Yao H, Carey KD, Dillon TJ, Stork PJS. Somatostatin activation of mitogen-activated protein kinase via somatostatin receptor 1 (SSTR1). Mol Endocrinol 1999; 13: Alderton F, Humphrey PPA, Sellers LA. High-intensity p38 kinase activity is critical for p21 induction and the antiproliferative function of Gi protein-coupled receptors. Mol Pharmacol 2001; 59: Matsushita H, Hara M, Endo Y et al. Proliferation of parathyroid cells negatively correlates with expression of parathyroid hormone-related protein in secondary parathyroid hyperplasia. Kidney Int 1999; 55: Cozzolino M, Lu Y, Finch J, Slatopolsky E, Dusso AS. p21waf1 and TGF-a mediate parathyroid growth arrest by vitamin D and high calcium. Kidney Int 2001; 60: