help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kifor, O.
Right arrow Articles by Brown, E. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kifor, O.
Right arrow Articles by Brown, E. M.
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4455-4464
Copyright © 2003 by The Endocrine Society

Decreased Expression of Caveolin-1 and Altered Regulation of Mitogen-Activated Protein Kinase in Cultured Bovine Parathyroid Cells and Human Parathyroid Adenomas

Olga Kifor, Imre Kifor, Francis D. Moore, Jr., Robert R. Butters, Jr., Thomas Cantor, Ping Gao and Edward M. Brown

Endocrine-Hypertension Division and Departments of Medicine (O.K., I.K., R.R.B., E.M.B.) and Surgery (F.D.M.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; and Scantibodies Laboratories (T.C., P.G.), Santee, California 92071

Address all correspondence and requests for reprints to: Dr. Olga Kifor, Endocrine-Hypertension Division, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: okifor{at}rics.bwh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Caveolins are key components of caveolae membranes. The calcium-sensing receptor (CaR) resides within caveolin-rich membrane domains in bovine parathyroid (PT) cells. Recent studies reported reduced CaR expression, and abnormal calcium-sensing in PT tumors. To examine this altered CaR signaling, we investigated ERK activation after CaR stimulation in human and bovine PT cells. In freshly prepared bovine PT cells, high extracellular calcium (Ca2+0) stimulates ERK1/2 phosphorylation, and activated ERK1/2 colocalizes with caveolin-1 at the plasma membrane but fails to translocate to the nucleus, and cell proliferation is low. In cultured bovine PT cells, CaR and caveolin-1 levels are reduced; activated ERK1/2 localizes in the cell periphery at 10 min and in the perinuclear and nuclear regions at 60 min after exposure to high Ca2+0, and cell proliferation is increased. In PT cells from adenomas, there are high levels of caveolin-2, variably reduced caveolin-1, and hyperactivation of ERK1/2, which colocalizes with caveolin-1 in some cells, but localizes in the cytosol and nucleus in others. Finally, caveolin-1 negative human PT cells exhibit reduced suppressibility of PTH secretion by high Ca2+0. Thus, CaR and caveolin-1 colocalize in PT cells, and reduced levels of caveolin-1 could participate in the abnormal cellular function and proliferation of cultured bovine PT cells and PT adenomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE G PROTEIN-COUPLED extracellular calcium (Ca2+0)-sensing receptor (CaR) was initially cloned from bovine parathyroid (PT) glands but has also been shown to be expressed in kidney, brain, and other tissues (1, 2). CaR activation by high Ca2+0 is thought to control the rate of PTH secretion from PT cells and the rate of calcium reabsorption by the kidney (2). The CaR couples to activation of phosphatidylinositol-specific phospholipase C, phospholipase A2 (PLA2), and phospholipase D and probably also to inhibition of adenylyl cyclase (2, 3). Mutations affecting the CaR that make it either less or more sensitive to Ca2+0 cause various clinical disorders (2, 4). There is abnormal regulation of PTH secretion by Ca2+0 in primary hyperparathyroidism both in vivo and in vitro (2, 4, 5). The abnormal Ca2+0-sensing in hyperparathyroidism has generally been attributed to decreased CaR expression (6, 7, 8, 9). The reduction of apparent CaR protein concentration may play a role in abnormal Ca2+0-regulated PTH release and, in turn, in the hypercalcemia observed in these hyperparathyroidism patients. Normal PT cells only rarely proliferate. The effect of Ca2+0 on PT cell growth is controversial (10, 11, 12, 13). Several lines of evidence support the hypothesis that the CaR plays a critical role in regulating PT cell proliferation (2, 4). In a rat fibroblast cell line, which expresses an endogenous CaR, high Ca2+0 induces cell proliferation by activating proliferation-associated signals, including ERK1/2 phosphorylation (14). In contrast, in PT cells the CaR seems to directly or indirectly mediate the inhibition of cell proliferation by high Ca2+0. Wada et al. (10) demonstrated that the calcimimetic compound NPS R-568 inhibits PT cell proliferation in rats with experimentally induced renal insufficiency. Abnormally increased PT cell proliferation is found uniformly in primary and secondary hyperparathyroidism.

Caveolae are plasma membrane organelles, characterized by a low solubility in Triton X-100, where specific lipid and protein components are subcompartmentalized (15). Caveolin, a 21- to 24-kDa integral membrane protein, is a principal component of caveolae membranes and exists as several isoforms (caveolin-1, -2, and -3) (15, 16). Caveolin-1 and caveolin-2 are found as long ({alpha}) and short (ß) isoforms lacking a few N-terminal amino acids, and they can produce large homo- and hetero-oligomers (232–443 kDa) consisting of 14–16 individual molecules (17, 18, 21). Caveolin may function as a scaffolding protein to organize and concentrate inactive signaling molecules within caveolae membranes for regulated activation by appropriate receptors and to facilitate cross talk between distinct signaling cascades. Caveolin-1 binding may also serve to terminate signal transmission after activation (16). Caveolin-1 interacts with and negatively regulates the activities of a number of signaling molecules, such as the {alpha}-subunit of G proteins, Ras, Src, nitric oxide synthase, growth factor, and G protein-coupled receptors, protein kinase C (PKC), and adenylyl cyclase (19, 20, 21, 22, 23, 24). A short cytosolic N-terminal region of caveolin is involved in the formation of oligomers and mediates the interaction of caveolin with these signaling molecules, which results in the inactivation of signaling (16).

Recent studies suggest that caveolae can modulate specific events that depend on calcium. Ca2+-ATPase, inositol 1,4,5-trisphosphate receptor-like protein, dihydropyridine-sensitive Ca2+ channels, and the CaR have all been localized to caveolae (25, 26, 27, 28). In addition, endothelial Ca2+ waves preferentially originate from caveolae (29), and Isshiki et al. (28) have shown that signal transduction from caveolae occurs in living cells and that caveolae may be a compartment involved in regulating store operated Ca2+ entry. Finally, absence of caveolae in cav-1 (-/-) null mice impairs calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone (30).

Targeted down-regulation of caveolin-1 is sufficient to drive cell transformation and hyperactivates the ERK kinase cascade (31, 32, 33). Coexpression with caveolin-1 dramatically inhibits signaling from epidermal growth factor receptor, Raf, MAPK kinase-1, and ERK-2 to the nucleus in vivo (33). Loss of caveolin-1 expression is predicted to lead to constitutive activation of the p42/44 MAPK cascade (31, 32, 33). In caveolin-1 (-/-) null mice, the MAPK cascade is, in fact, hyperactivated (34, 35). Many G protein-coupled receptors have been shown to induce cellular growth or differentiation responses through activation of the MAPK cascade (36). MAPKs are able to phosphorylate multiple substrates that are found in various subcellular locations: membrane-associated, such as the epidermal growth factor receptor (36); cytoplasmic, such as c-PLA2 (36, 37); or nuclear, such as Ets-like protein-1 (Elk-1) and Sap-1a (38, 39). Phosphorylation of Elk-1 increases its affinity for the serum-response factor and enhances transcription of growth-related proteins, such as c-Fos (39). Prevention of MAPK nuclear translocation strongly inhibits Elk-1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors (39).

Previously, we showed that bovine PT cells express caveolin-1, and that the CaR resides within caveolae (26). Other signaling proteins, including Gq/11, endothelial nitric oxide synthase, glycosylphosphatidylinositol-anchored alkaline phosphatase, and several PKC isoforms ({alpha}, {delta}, and {zeta}), are also present in PT caveolae, and activation of the CaR by high Ca2+0 increases tyrosine phosphorylation of caveolin-1 (26). Here, we address the possible role of caveolin-1 and alterations in its expression in CaR-mediated signal transduction in cultured bovine PT cells as well as PT adenomas. Because caveolin-1 is thought to be a negative regulator of signal transduction and proliferation, the reduction of caveolin-1 in cultured bovine PT cells and human PT adenomas may participate in the deranged cellular function of these cell types.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Monoclonal antibodies to caveolin-1 (C73120) and caveolin-2 (C57820) were purchased from BD Transduction Laboratories (Lexington, KY). Polyclonal antibody and blocking peptide to caveolin-1 and -2, and protein A/G agarose beads were obtained from Santa Cruz Biochemicals (San Francisco, CA). A monoclonal antibody and a polyclonal antiserum against phosphorylated ERK1/2 [anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody] and an antibody against Elk-1 and phosphorylated Elk-1 (pElk-1) were purchased from New England Biolabs (Beverly, MA). CaR-specific polyclonal antisera, which were raised in rabbits against peptides based on the CaR sequence (antiserum 4637 to amino acids 345–359), and CaR-transfected human embryonic kidney 293 (HEKCaR) cells were generous gifts of NPS Pharmaceuticals, Inc. (Salt Lake City, UT). Peroxidase-conjugated, goat antimouse and antirabbit antibodies were from Sigma (St. Louis, MO). Goat antimouse IgG coupled to Alexa-488 and goat antirabbit IgG coupled to Alexa-568 were purchased from Molecular Probes, Inc. (Eugene, OR). A PTH-specific sheep antiserum (GW-1) was prepared in our laboratory and recognizes intact PTH as well as carboxyterminal fragments of the hormone (40).

Methods

Cell preparation and incubation. Bovine and human PT cells were prepared by collagenase and deoxyribonuclease digestion from PT glands as previously described (3, 37). Cell viability was determined by trypan blue exclusion and routinely exceeded 95%. The cells were used immediately as acutely dispersed cells or were cultured in multiwell plates in DMEM Ham’s F-12 or medium 199 (GIBCO-BRL, Gaithersburg, MD) with 10% fetal calf serum, 5 µg/ml insulin, and antibiotics (3, 37). For immunocytochemistry, the cells were cultured on glass coverslips. The ionized calcium concentration in the medium was about 0.5 mmol/liter. More than 95% of the dispersed cells consisted of typical PT cells, as revealed by immunostaining with an anti-PTH antibody. Before cell plating, the culture wells and coverslips were coated with fibronectin to enhance cellular attachment. Growth of PT cells in culture was initiated from clumps of cells that rapidly attached to the culture plate and gradually spread out. The morphological appearance of these PT cells was typically round or polygonal; spindle-shaped cells—presumably fibroblasts—were present as well, and comprised 5% of the cells after 2–10 d in culture. HEK-293 cells stably transfected with the human CaR cDNA (HEKCaR cells) were grown on glass coverslips or in 24-well plates in DMEM (not containing sodium pyruvate) with 10% fetal calf serum. Subconfluent monolayers of PT cells and HEKCaR cells were serum-starved for 24 h before incubation with varying levels of Ca2+0.

Immunocytochemistry. Coverslips with adherent PT cells or HEKCaR cells were washed with ice-cold, PBS and fixed for 10 min with 4% formaldehyde in PBS at room temperature. The slides were then incubated for 10 min with an inhibitor of endogenous peroxidase (Dako Corp., Carpinteria, CA), or with 0.1 mol/liter glycine (pH 7.4) to suppress autofluorescence. To reduce nonspecific binding, this step was followed by incubation for 30 min with blocking solution (1% BSA and 1% normal goat serum in PBS). The slides were subsequently incubated with first antibodies in blocking solution overnight in a humidified chamber at 4 C. After washing, the slides were incubated with the appropriate peroxidase-conjugated secondary antibodies, and immunostaining was visualized with the Dako AEC Substrate System (Dako Corp.) as described (7, 40). For double immunofluorescence staining, the slides were incubated overnight with a mixture of first antibodies. The next day, the preparations were washed and incubated with a mixture of secondary antibodies tagged with Alexa 488 or Alexa 568 in PBS containing 1% BSA for 30 min at 37 C. The slides were then washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA) to prevent photobleaching. Fluorescence images were obtained using the x100 objectives of a Bio-Rad (Hercules, CA) MRC 1024/2P multiphoton microscope equipped with Krypton and Argon lasers at the Brigham and Women’s Hospital Confocal Microscopy Core facility. The images were collected sequentially with the use of selected excitation wavelengths. LaserSharp software (Bio-Rad) permitted the collection of a series of XY sections at specific increments (0.35 or 0.5 µm) in the Z direction. Each of these image files was converted to montages and/or auto montages. Alexa-568 produced red and Alexa-488 produced green signals. The red and green components were merged, and the composite images as well as the individual red and green images are presented in some cases. The autofluorescence of the samples was minimal and was subtracted from the values obtained during measurements.

Electrophoresis and immunoblots. Western blot analysis was performed essentially as previously described (26, 37). The cells were rinsed with ice-cold PBS, and lysed in ice-cold lysis buffer containing: 10 mmol/liter Tris-HCl (pH 7.4), 1 mmol/liter EDTA, 1.0 mmol/liter EGTA, 0.25 mol/liter sucrose, 1% Triton X-100, and protease inhibitors (10 µg/ml each of aprotinin, leupeptin, calpain inhibitor, and 100 µg/ml Pefabloc). Nuclei and cell debris were removed by low-speed centrifugation (1000 x g for 10 min), and the resultant cell lysate in the supernatant was used for Western blot analysis. Equal amounts of proteins were mixed with 2x sodium dodecyl sulfate (SDS)-Laemmli buffer containing 100 mmol/liter dithiothreitol, separated on 7.5 or 10% SDS-PAGE or on linear 4–10% SDS gradient gels, and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The blots were incubated with blocking solution (PBS with 0.25% Triton X-100 and 5% nonfat dry milk) for 1 h at room temperature. The membranes were then incubated overnight with primary antiserum and, after washing, with secondary antibodies. The bands were visualized by chemiluminescence (Renaissance ECL system, NEN Life Science Products, Boston, MA). Protein concentration was determined with a Micro BCA protein kit (Pierce Chemical Co., Rockford, IL). For determination of ERK1/2 phosphorylation, cells were lysed directly in boiling sample buffer, and proteins were resolved by SDS-PAGE.

Immunoprecipitation. Cells were washed with ice-cold PBS and lysed with immunoprecipitation buffer containing 150 mmol/liter NaCl, 10 mmol/liter Tris (pH 7.4), 1% glycerol, 1 mmol/liter EDTA, 1 mmol/liter EGTA, 1 mmol/liter sodium O-vanadate, protease inhibitors (as described above), N-octyl-ß-D-glucopyranoside 60 mmol/liter, and 1% Triton X-100 (26). The cell lysates were centrifuged at 10,000 x g for 10 min. Supernatant proteins (300 µg of total lysate of human PT cell samples, and 150 µg of bovine PT cell lysate) were incubated with 2–4 µg of monoclonal antibodies to caveolin-1 or with irrelevant mouse IgG for 1 h at 4 C. Protein A/G-agarose beads were then added for a further 1 h at 4 C. Bound immunocomplexes were washed, and the pellets were eluted by boiling for 5 min with 2x Laemmli sample buffer, and after SDS-PAGE, Western blot analysis was performed, as described (26, 37).

Purification of caveolin-rich membrane fractions. Fractions enriched in caveolin were purified as described previously (17, 34). Tissues or cells were homogenized with 2 ml 2-(N-morpholino)-ethanesulfonic acid-buffered saline (MBS) containing 25 mmol/liter 2-(N-morpholino)- ethanesulfonic acid (pH 6.5), 0.15 mol/liter NaCl, protease inhibitors, and 1% Triton X-100. The homogenate was then adjusted to 40% sucrose by the addition of an equal volume of 80% sucrose prepared in MBS. The mixture was placed on the bottom of an ultracentrifuge tube, overlaid with a discontinuous 5–35% sucrose gradient in MBS and sedimented at 38,000 rpm for 18 h in an SW40 rotor (Beckman, Palo Alto, CA). Fractions (1.3 ml) were removed sequentially from the top of the gradient and designated as fractions 1–9 (fractions 2–3 and 6–9 are considered to be of caveolar Triton X-insoluble, and Triton X-100-soluble, noncaveolar origin, respectively). Equal amounts of proteins from each of the fractions were subjected to SDS-PAGE and immunoblotting.

Determination of Ca2+0-regulated PTH release. Dispersed human PT cells (1 x 106 cells/0.5 ml) were incubated with 0.5 mmol/liter Mg2+0 and varying concentrations of Ca2+0 (0.5–2.0 mmol/liter) for 1 h at 37 C in Eagle’s MEM with 2% normal human serum (5). Supernatant PTH was measured using the Whole PTH (1–84) Specific Immunoradiometric Assay kit (Scantibodies Laboratory, Inc., Santee, CA) (41).

Analysis of cellular proliferation. Primary cultures of bovine PT cells were seeded in 96-well plates at a density of 10,000 cells/well at d 0 in quadruplicate. After 2 and 10 d, 5-bromo-2'-deoxy-uridine (BrdU) incorporation was assessed with the use of the Cell Proliferation ELISA/BrdU (fluorescent) kit from Roche Diagnostic (Indianapolis, IN), according to the manufacturer’s recommendations.

Statistics

Data presented are mean ± SEM of the indicated number of experiments. The Sigmastat software (SPSS, Chicago, IL) was used to analyze the results. One-way ANOVA with Student-Newman-Keuls test was used. A P value of less than 0.05 was considered to indicate a statistically significant difference.

The studies using human PT adenomas were carried out under a protocol approved by the local institutional review board for the use of discarded human materials.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reduced immunostaining for caveolin-1 in PT cells prepared from human PT adenomas

To determine whether caveolin-1 is present in human pathologic PT cells, we performed immunohistochemical analysis of frozen sections of PT glands. Figure 1AGo shows a rim-like distribution of caveolin-1 positive PT cells—presumably a rim of normal PT tissue—around the periphery of a section from an adenoma. The immunostaining for caveolin-1 in these cells was considerably more intense than in the chief cells of the adenoma in the left hand portion of Fig. 1AGo or the chief cells in another adenoma shown in Fig. 1BGo. In other adenoma sections in Fig. 1CGo, there was distinct staining of cells for caveolin-1, which was essentially abolished by preabsorption of the first antibody with the peptide against which it was raised (Fig. 1DGo). Figure 1EGo shows a representative example of the immunostaining for caveolin-2 of another PT adenoma, which was abolished by preabsorption with specific peptide (Fig. 1FGo).



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 1. Reduced immunostaining for caveolin-1 (Cav)-1 in human PT (HPT) adenomas. Frozen sections taken from HPT adenomas were immunostained with monoclonal antibody to Cav-1 and subsequently incubated with peroxidase-coupled goat antimouse secondary antibody as described in Materials and Methods. A, A rim of cells around the periphery and isolated groups of cells within the adenoma stain considerably more intensely than do the majority of the chief cells in the left hand portion of A or those in another adenoma section in B. Comparison of the immunostaining of HPT adenomas using polyclonal antibodies to caveolin-1 (C and D) or to Cav-2 (E and F), without (C and E) or after preincubation of the antibodies with the specific peptides against which they were raised (D and F). G, Representative immunoblot analysis showing the relative levels of expression of Cav-1 and Cav-2 in bovine PT cells (BPT), in HPT cells prepared from PT adenomas and in HEKCaR cells using polyclonal antibodies to Cav-1, and Cav-2 without or after preincubation of the antibodies with the specific peptides. Chemiluminescence detection was used in samples of whole-cell lysates (20 µg/lane) immunoblotted with antibodies to Cav-1 and Cav-2 as described in Materials and Methods.

 
Next, the presence of caveolin-1 and caveolin-2 were determined by Western blot analysis in human PT cells prepared from PT adenomas, bovine PT cells, and HEKCaR cells. A representative immunoblot shows the relative levels of caveolin-1 and -2 in 18 PT adenomas in Fig. 1GGo. Note that caveolin-2 is abundantly expressed in HEKCaR cells and in all human PT adenomas examined. The caveolin-2 immunoblot showed some caveolin-2 immunoreactive species of higher or lower molecular masses, which are probably the {alpha}, ß, and {gamma} isoforms of caveolin-2. Figure 1GGo also illustrates that substantial caveolin-1 is expressed in some human PT glands but not in others. Western blots were subjected to densitometric analysis, and integral OD was determined for the caveolin-1 protein band in 52 adenomas studied in this way (data not shown). Quantitative determination of the caveolin-1 immunoreactivity showed that caveolin-1 is present at substantial levels in 22 cases (+; 100–800 OD), whereas lower but clearly detectable levels of caveolin-1 are present in 10 cases (+/-; 10–100 OD). No immunoreactivity (-) was detected in 20 glands, probably because the caveolin-1 levels are very low and could not be detected by Western blot analysis in 20-µg protein samples.

CaR associates with caveolin-rich protein fractions in human PT cells

Caveolins have been found to segregate into detergent-insoluble membrane domains. Human PT cells were homogenized and fractionated, and Triton X-100 insoluble and soluble fractions were immunoblotted with antibodies to CaR, caveolin-1, -2, -3, and flottilin. Figure 2AGo shows that most of the caveolin-1, caveolin-2, flotillin (another protein associated with caveolae-like structures), and CaR protein were concentrated in detergent-insoluble caveolar fractions (2, 3, 4) prepared from a caveolin-1-expressing PT adenoma (patient 18). Immunostaining for caveolin-3 was negative (data not shown). Finally, to confirm the stable interaction between the CaR and caveolin-1 that we have previously observed in bovine PT cells (26), we performed immunoprecipitation experiments. Cell lysates were prepared from bovine and human PT cells (patient 18), immunoprecipitated with caveolin-1 antibody, and then subjected to Western blot analysis with CaR antiserum. As shown in Fig. 2BGo, caveolin-1 coimmunoprecipitates with the CaR in bovine PT and in caveolin-1-expressing human PT cells. The use of nonspecific mouse IgG shows no such immunoprecipitation (negative control), and Western blotting of HEKCaR membranes that were not immunoprecipitated serves as a positive control for the CaR.



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 2. Association of caveolin (Cav) with CaR in human PT (HPT) cells. HPT cell lysates were prepared from the adenoma of patient 18 (A) and were subjected to subcellular fractionation after homogenization in a buffer containing 1% Triton X-100 (see Materials and Methods). Samples of caveolar fractions were immunoblotted with monoclonal antibodies to Cav-1, Cav-2, or flotillin, or a polyclonal antibody to the CaR (4637). Results are representative of those obtained in three separate preparations. B, Bovine PT (BPT) and HPT (patient 18) cell lysates were prepared and immunoprecipitated (IP) with monoclonal antibodies to caveolin-1, or HPT lysates were immunoprecipitated with nonspecific mouse IgG (IgGm), as detailed in Materials and Methods. Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblot analysis for the CaR with 4637 antiserum. HEKCaR represents a positive control for the CaR using membranes prepared from CaR-transfected HEK293 cells without immunoprecipitation. Results are representative of those obtained in two separate preparations.

 
CaR is localized in caveolae on the PT cell surface

The subcellular localization of the CaR and caveolins in bovine and human PT cells and in HEKCaR cells was studied by indirect immunofluorescence with a confocal microscope. There was clear colocalization of the CaR with caveolin-1 at the plasma membrane in bovine PT cells as indicated by yellow staining—some in the form of dots—in the merged images (Fig. 3Go, A–C). A tight correlation between the intensity of green and red fluorescence and the resultant yellow in the merged image (Fig. 3MGo) suggests the existence of macromolecular complexes containing both caveolin-1 and the CaR. As shown in Fig. 3Go, D–F, there was substantial overlap in the staining for caveolin-1 and the CaR in human PT cells prepared from patient 18, whereas minimal staining was present in cells prepared from the PT gland of patient 16 (Fig. 3Go, G–I and N). In HEKCaR cells, caveolin-2 shows a Golgi-like distribution and colocalizes with the CaR in this region; it produces minimal signals at the plasma membrane except for scattered yellow dots in the merged image indicating colocalization with the CaR (Fig. 3Go, J–L and O).



View larger version (61K):
[in this window]
[in a new window]
  		FIG. 3. Immunofluorescent detection of caveolin and CaR proteins in bovine and human PT cells and in HEKCaR cells. Dispersed bovine PT cells (A–C), human PT cells (D–I) and HEKCaR cells (J–L) were fixed on coverslips, and stained for the CaR with polyclonal rabbit antibody (A, D, G, and J) for caveolin-1 (B, E, and H), and for caveolin-2 (K) with monoclonal antibodies as detailed in Materials and Methods. The distributions of the CaR and caveolins were visualized by fluorescence and colocalization analysis. C, Merged image analysis of A and B; F, merged image of D and E; I, merged image analysis of G and H; and L, merged image of J and K. M, Correlation between the two fluorophores used for A and B; N, correlation between G and H; and O, correlation between J and K. Magnification, x1000.

 
Reduction of caveolin-1 and CaR in cultured bovine PT cells

Reduced expression of the CaR has been demonstrated in bovine PT cells maintained in culture for several days (7, 8, 9). Therefore, we investigated whether this reduction of CaR expression in cultured bovine PT cells was also associated with a reduction of the caveolin-1 protein. Cell surface staining for both the CaR and caveolin-1 decreased markedly after 2–5 d in culture (Fig. 4Go).



View larger version (120K):
[in this window]
[in a new window]
  		FIG. 4. Progressive reduction of CaR and caveolin-1 in cultured bovine PT cells. Acutely dispersed cells (A and D) or cells cultured for 2 (B and E) or 5 d (C and F) were fixed with 4% formaldehyde. After blocking, the cells were immunostained with antibodies to the CaR (A–C) or to caveolin-1 (D–F) and subsequently with peroxidase-linked secondary antibodies. The photomicrographs were taken at a magnification of x400. Note that the cells at d 0 stain considerably more intensely than do those at 2 or 5 d.

 
Phosphorylation of ERK1/2 in bovine and human PT cells

Previously, we observed that high Ca2+0 stimulates ERK1/2 phosphorylation in acutely dispersed bovine PT cells and in HEKCaR cells (37). Recently, it has been suggested that caveolin-1 is a negative regulator of a variety of MAPK cascades (16). Loss of caveolin-1 expression is predicted to lead to constitutive activation of the MAPK cascade (16). Therefore, we investigated whether the presence of caveolin-1 or a reduced level of caveolin-1 was associated with any effect on ERK1/2 phosphorylation in PT cells. Figure 5Go shows that, whereas phosphorylation of ERK1/2 was increased at high but not at low Ca2+0 in freshly prepared bovine PT cells and in HEKCaR cells, phosphorylated ERK1/2 (pERK1/2) was increased at both low and high Ca2+0 in bovine PT cells cultured for 10 d. In freshly prepared human PT cells, there was considerable variation in activation of ERK1/2 by low and high Ca2+0 with most preparations showing equivalent levels at 0.5 and 3.0 mmol/liter Ca2+0. Cell prepared from patient 42 showed significantly increased ERK1/2 phosphorylation in the presence of 3.0 mmol/liter Ca2+0 relative to that observed at 0.5 mmol/liter Ca2+0.



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 5. ERK1/2 phosphorylation in bovine and human PT (HPT) cells and in HEKCaR cells. Dispersed bovine PT cells (BPT) (on d 0 or cultured for 10 d), HEKCaR cells and dispersed HPT cells were incubated with 0.5 or 3.0 mmol/liter Ca2+0 in standard medium containing 0.2% BSA for 10 min. pERK1/2 was measured by Western blot analysis with a phospho-ERK1/2-specific antiserum as described in Materials and Methods.

 
Intracellular localization of endogenously activated ERK1/2 in bovine and human PT cells

We analyzed the subcellular distribution of CaR-activated pERK1/2 at high (3.0 mM) Ca2+0 by indirect immunofluorescence and compared it with the distribution of caveolin-1 and nonphosphorylated or phosphorylated Elk-1 (Fig. 6Go). In nonstimulated bovine PT cells (0.5 mmol/liter Ca2+0), only minimal immunostaining was observed for activated ERK1/2 (data not shown—similar in appearance to Fig. 6HGo). Treatment of bovine PT cells and human PT cells prepared from a caveolin-1 positive PT adenoma with 3.0 mmol/liter Ca2+0 for 10 min resulted in the appearance of activated ERK1/2 (Fig. 6Go, B and E) in plasma membrane caveolae (Fig. 6Go, A and D) with minimal localization in the nucleus. Merged images prove the colocalization of the pERK1/2 with caveolin-1 (yellow color in Fig. 6Go, C and F). After 1 h incubation of bovine PT cells with 3.0 mmol/liter Ca2+0, we observed a significant reduction of activated ERK1/2 (Fig. 6HGo) without translocation to the nuclear compartment as indicated by the location of Elk-1 in red (Fig. 6Go, G and I) without any detectable colocalization of pERK1/2 and Elk-1 (I, GH). After 10 d of culture, bovine PT cells, in contrast, exhibited some degree of colocalization of phosphorylated Elk-1 and ERK1/2 (small yellow spots in Fig. 6LGo) in the cytosol and the nucleus after incubation for 60 min at 3.0 mmol/liter Ca2+0. Nuclear localization of pERK1/2 was seen after 1 h incubation with 3.0 mmol/liter Ca2+0 in human PT cells as indicated by some degree of colocalization of pERK1/2 and pElk-1, as was seen with cultured bovine PT cells incubated with 3.0 mM Ca2+0 for 60 min.



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 6. Colocalization of pERK1/2 with caveolin-1 or with Elk-1 or pElk-1. Freshly prepared bovine PT cells (A–C) and human PT cells (D–F) were incubated with 3.0 mmol/liter Ca2+0 for 10 min. After fixation and permeabilization, they were immunostained with polyclonal antibody to caveolin-1 (A and D) and subsequently with a secondary antibody tagged with Alexa 568 (red); and for pERK1/2 with a monoclonal antibody (B and E) and subsequently with a secondary antibody tagged with Alexa 488 (green). The superimposed images (yellow) show colocalization of caveolin-1 with pERK1/2 (C and F). There was a tight correlation between caveolin-1 and pERK1/2 in panels A-B and D-E. Freshly prepared bovine PT cells (G–I) were incubated with 3.0 mmol/liter Ca2+0 for 1 h and immunostained for Elk-1 (G) and for pERK1/2 (H). Ten-day cultured bovine PT cells (J–L) and freshly prepared human PT cells (M–O) were incubated with 3.0 mmol/liter Ca2+0 for 1 h and immunostained for pElk-1 (J and M) and for pERK1/2 (K and N). For detection of pERK1/2 secondary antibody tagged with Alexa 488 (green), and for detection of Elk-1 and pElk-1 secondary antibody tagged with Alexa 568 (red) were used. Images H and I show no significant immunostaining for pERK1/2. Images in L and O show localization of pERK1/2 and pElk-1 in cytosol and in the nuclear compartment. Images AB, DE, GH, JK, and MN show the correlation between the red and green colors, and yellow color along the 45-degree line represents areas of the images of the cells in which there is colocalization of the red and green images.

 
Filipin inhibits high Ca2+0-induced activation of ERK1/2 in bovine PT cells

Filipin, a sterol-binding agent, binds to cholesterol (a major component of caveolae) and disrupts caveolar structure and function (32). To investigate whether the CaR-stimulated, MAPK phosphorylation cascade originates in PT cell caveolae, we examined the effects of filipin on this process. Figure 7Go shows that pretreatment of acutely dispersed bovine PT cells with filipin inhibited high Ca2+0-induced activation of ERK1/2 in a dose-dependent manner, whereas it had no effect on high Ca2+0-induced ERK1/2 phosphorylation in HEKCaR cells, which lack caveolin-1.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 7. Filipin-mediated inhibition of ERK1/2 activation in bovine PT cells. Bovine PT cells and HEKCaR cells were treated with increasing concentrations of filipin for 1 h at 37 C, then incubated for an additional 10 min with 3.0 mmol/liter Ca2+0. ERK1/2 phosphorylation (pERK1/2) was measured by Western blotting with a phospho-ERK1/2-specific antiserum. Integral optical densities were determined and normalized to the values observed at 3.0 mmol/liter Ca2+0 (8 ). The sum of pERK1 and pERK2 is shown. Data represent the mean ± SEM for three and two separate preparations in duplicate for PT cells vs. HEKCaR cells. pERK1/2 was significantly inhibited by 3 and 5 µg/ml filipin in bovine PT cells (P < 0.05).

 
Presence of caveolin-1 is associated with increased responsiveness of PTH release to elevated Ca2+0 concentrations in dispersed human PT cells

The effect of increasing concentrations of Ca2+0 on PTH release was investigated in cells obtained from 28 adenomas (Fig. 8Go). By examining the presence of caveolin-1, the glands segregated into three major groups. Group A comprised caveolin-1 positive (+) cells obtained from 12 adenomas, and group B contained cells prepared from four adenomas that were less positive (±) for caveolin-1. Finally, a third group of cells (group C) prepared from 12 adenomas was negative (-) for caveolin-1. We observed that incubation with a high Ca2+0 concentration had less suppressive effect on PTH release in groups B and C. In cells prepared from caveolin-1 negative adenoma or those with low levels of caveolin-1, 2.0 mmol/liter Ca2+0 inhibited PTH release by only about 20%. Caveolin-1 containing PT cells in group A, in contrast, displayed approximately 45% inhibition of PTH release at 2.0 mmol/liter Ca2+0 (from 1218 ± 76 to 652 ± 65 pg/500,000 cells). This represents a significant inhibition of PTH release (P < 0.05) by high Ca2+0 in group A. The mean of PTH concentration ranged from 1272 ± 125 to 1032 ± 117 pg/500,000 cells in group C at 0.5 mmol/liter Ca2+0 to 2.0 mmol/liter Ca2+0. There was no significant difference in PTH release at 0.5 and 2.0 mmol/liter Ca2+0 in this group. PTH release at high Ca2+0, on the other hand, was significantly less (P < 0.05) in group A than in group C.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 8. Inhibition of PTH release from human PT cells in the presence of different Ca2+0 concentrations. Dispersed human PT cells (1 x106 cells/0.5 ml) were prepared from 28 adenomas and were incubated with 0.5 mmol/liter Mg2+0 and varying concentrations of Ca2+0 (0.5–2.0 mmol/liter) for 1 h at 37 C in Eagle’s MEM with 2% normal human serum. Supernatant samples from triplicate incubation vials were collected and assayed for PTH using the whole immunoradiometric assay specific for PTH (1–84). Data represent the mean ± SEM for three to six observations at each Ca2+0 concentration in one experiment. PTH release at 1.5 mmol/liter Ca2+0 in group A was significantly less than that at 1.5 mmol/liter Ca2+0 (P < 0.05) in groups B and C. PTH release at 2.0 mmol/liter Ca2+0 in group A was significantly less than that at 2.0 mmol/liter Ca2+0 in groups B and C. The absolute values for PTH release at 0.5 mmol/liter Ca2+0 for groups A, B and C were 1218, 1119, and 1272, respectively.

 
Bovine PT cell proliferation

Transgenic expression of caveolin-1 in mouse embryonic fibroblasts is responsible for initiating growth arrest (43). Caveolin-1 induces cells to exit the S-phase of the cell cycle, with a concomitant increase in the G0/G1 population and reduction of cellular proliferation (43). To assess whether caveolin-1 expression is associated with a reduction in the S-phase population in bovine PT cells, we used BrdU incorporation (Fig. 9Go). In association with the decrease in the level of caveolin-1 protein in cultured bovine PT cells (see Fig. 4Go), cell number (data not shown) and BrdU incorporation increased in bovine PT cells cultured for 10 d as compared with cells after 1–2 d culture. The fibroblasts were less than 5% in our primary cultures maintained to 10 d. There was no significant difference in PT cell number (data not shown) or in BrdU incorporation at 0.5 and 3.0 mmol/liter Ca2+0 in any of the cells studied.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 9. BrdU incorporation. Bovine PT (BPT) cells were plated in 96-well plates at a density of 10,000 cells/well at d 0. After 2 or 10 d, BrdU incorporation was determined as described in Materials and Methods. Data represent the mean ± SEM for two separate preparations in quadruplicate. The BrdU incorporation significantly increased in adherent PT cell layer after 10 d in culture (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Abnormal regulation of PTH secretion by Ca2+0 contributes importantly to the pathophysiology of hypercalcemia in primary hyperparathyroidism (2, 5). The abnormal PT cells secrete more PTH than normal PT cells at a given level of Ca2+0. Recent studies have shown that in most cases of primary hyperparathyroidism, there is a reduced sensitivity to Ca2+0 (e.g. an increase in set-point) (2, 5), the mechanisms for which have not been established with certainty. Reduced expression of the CaR has been demonstrated in pathological PT glands of patients with primary and secondary hyperparathyroidism (6, 7, 8, 9). Corbetta et al. (9) observed that the levels of Gq/11 were lower in the majority of pathological PT tissue samples than in normal control tissue. Previously, we showed that the CaR is located primarily in bovine PT caveolae in association with Gq/11 and PKC (26). In this study, we show that human PT cells prepared from adenomas express caveolin-2 and flotillin, and show a variable degree of reduction in caveolin-1. Only 62% of the PT adenomas studied here expressed caveolin-1, as assessed by Western blot analysis, and, in general, the cells expressing higher levels of caveolin-1 displayed greater suppressibility of PTH release by high Ca2+0. Conversely, lower levels of caveolin-1 expression or the absence of caveolin-1 was associated with a higher degree of resistance to Ca2+0 in most cases. It is possible, therefore, that CaR localization in caveolae plays a role in Ca2+0-regulated PTH release. Decreased expression of caveolin-1 associated with reduced signaling molecules (e.g. G proteins) could produce altered regulation of PTH release.

Caveolin-1 is a potent negative regulator of a variety of mitogenic signaling pathways (16). The loss of caveolin-1, but not of caveolin-2, has been observed in several tumor-derived cell lines (45, 46). To address whether caveolins influence CaR signaling, we analyzed the localization of the MAPK cascade in bovine PT cells. Previously, we observed that elevating Ca2+0 elicited rapid, dose-dependent phosphorylation of ERK1/2 in bovine PT and HEKCaR cells (37). Cellular responses to extracellular signals require coordinated control. In the case of ERK1/2, the mechanisms underlying not only its activation but also its dephosphorylation and inactivation are of considerable interest, because sustained ERK activation is important for proliferative signaling (36). In HEKCaR cells, 3.0 mM Ca2+0 stimulation produced a 17-fold increase in activated ERK1/2 at 10 min, which declined after 30–60 min, but persisted at a level above baseline for as long as 24 h (37). Caveolin-1 is a natural endogenous inhibitor of ERK (31, 32). Indeed, in freshly prepared bovine PT cells, which express a high level of caveolin-1, high Ca2+0-stimulated ERK1/2 phosphorylation peaked at 10 min, producing a 7-fold increase in activity, decreased to the basal level after 1 h, and then remained at that level (37). Now, we have shown that activated pERK1/2 colocalizes with caveolin-1 at the plasma membrane, and the amount of pERK1/2 decreased substantially after 1-h incubation with high Ca2+0 without translocation to the nuclear compartment. In addition, selective disassembly of caveolae with filipin impairs CaR-induced ERK1/2 phosphorylation in bovine PT cells but not in HEKCaR cells, which lack caveolin-1 (23). Thus this study reveals that structural integrity of caveolae is necessary for early activation and termination of the MAPK cascade in bovine PT cells. In human normal PT cells, high Ca2+0 induced activation of ERK1/2, and PD98059, a specific MAPK kinase inhibitor, abolished the inhibitory effect of 1.5 mmol/liter Ca2+0 on PTH release (47). In human PT cells, prepared from adenomas there was considerable variation in activation and localization of activated ERK1/2. In the majority of adenomas, we observed constitutively activated ERK1/2-independent of Ca2+0. In cells expressing high levels of caveolin-1, we observed that the high Ca2+0 activated pERK1/2 colocalized with caveolin-1 in the plasma membrane caveolar compartment, in other cells with reduced levels or lack of caveolin-1, pERK1/2 localizes in the plasma membrane, cytosol, and nucleus.

Bovine PT cells maintained in culture exhibit a rapid and marked (up to 89%) reduction in their levels of CaR mRNA and protein and gradually lose their responsiveness to Ca2+0 (11, 12, 13, 40, 42). The allosteric CaR activator, NPS R-467, has been shown to activate ERK1/2 phosphorylation and to inhibit both PTH secretion and PT proliferation (10, 37). Culture conditions do not usually permit the maintenance of functionally active bovine PT cells for long periods (11, 12, 13). Nygren et al. (13) suggested that bovine PT cells functionally dedifferentiate in culture conditions to a state similar to that of hyperparathyroidism. Indeed, in cultured bovine PT cells, we have observed reductions of CaR, high Ca2+0-induced stimulation of phosphatidylinositol-specific phospholipase C, and PLA2, as well as loss of responsiveness of PTH secretion to Ca2+0 (3, 40), and we have now likewise demonstrated reduction of caveolin-1 and hyperactivation of ERK1/2. Loss of caveolae in bovine PT cells could be responsible for impaired metabolic functions. Because caveolae in bovine PT cells are known to contain such signaling molecules as CaR, Gq/11, endothelial nitric oxide synthase, and several PKC isoforms, the loss of caveolin-1 expression in cultured bovine PT cells may indicate a reduced signal transduction capacity as part of the dedifferentiation process noted by Nygren et al. (13).

Caveolin-1 protein expression is highest in terminally differentiated cells, which are in the G0 phase of the cell cycle (43). Cyclins are small proteins that determine progression through the cell cycle. Overexpression of cyclin D1 is found in 20–40% of human PT adenomas (48). The cyclin D1 (PRAD1) oncogene is rearranged with the PTH gene and is transcriptionally activated in a subset of PT adenomas (49). Overexpression of cyclin D1 (PRAD1) induced by the PTH gene rearrangement is thought to be one of the genetic abnormalities responsible for tumorigenesis in sporadic primary PT adenomas (49). Cyclin D1 protein promoter activity was selectively repressed by caveolin-1, which required the caveolin-1 N terminus (43, 50). Caveolin-1 can inhibit cell proliferation via cell-cycle arrest in the G0/G1 phase (43, 50). Thus the reduction of caveolin-1 protein expression observed in human PT cells may contribute to increased cyclin D1 activity and the associated PT cell hyperplasia in some cases of PT adenoma. In pathological human PT tissue, there may be parallel abnormalities in the control of PTH secretion and PT cellular proliferation similar to those in cultured bovine PT cells. This down-regulation of caveolin-1 may contribute to adenomatous PT cell proliferation and function.

In conclusion, these data suggest the functional role of compartmentalized signaling in caveolin-1-expressing PT cells, and the potential contribution of down-regulation of caveolin-1 in the deranged Ca2+0-regulated PTH secretion and proliferation of cultured bovine and human PT cells prepared from adenomas.


   Acknowledgments
 
We thank Dr. Phillip Allen and Michelle Lowe for expert assistance with confocal imaging. The Whole PTH (1–84) Specific Immunoradiometric Assay kit was generously provided by Scantibodies Laboratories, Inc. (Santee, CA).


   Footnotes
 
This work was supported by generous grants from the NIH (DK41415, 48330, and 52005), NPS Pharmaceuticals, and the St. Giles Foundation.

Abbreviations: BrdU, 5-Bromo-2'-deoxy-uridine; Ca2+0, extracellular calcium; CaR, calcium-sensing receptor; Elk-1, Ets-like protein-1; HEK293, human embryonic kidney (cells); HEKCaR, CaR-transfected HEK293 cells; MBS, 2-(N-morpholino)-ethanesulfonic acid-buffered saline; pElk-1, phosphorylated Elk-1; pERK1/2, phosphorylated ERK1/2; PKC, protein kinase C; PLA2, phospholipase A2; PT, parathyroid; SDS, sodium dodecyl sulfate.

Received September 11, 2002.

Accepted May 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brown EM, Gamba G, Riccardi D, Lombardi D, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC 1993 Cloning and characterization of an extracellular Ca2+0-sensing receptor from bovine parathyroid. Nature 366:575–580[CrossRef][Medline]
  2. Brown EM, Vassilev PM, Quinn S, Hebert SC 1999 G-protein-coupled, extracellular Ca2+0-sensing receptor: a versatile regulator of diverse cellular functions. Vitam Horm 55:1–71[Medline]
  3. Kifor O, Diaz R, Butters R, Brown EM 1997 The Ca2+-sensing receptor (CaR) activates phospholipase C, A2, and D in bovine parathyroid and CaR-transfected human embryonic kidney (HEK293) cells. J Bone Miner Res 12:715–725[CrossRef][Medline]
  4. Brown EM, Kifor O, Bai M 1999 Decreased responsiveness to extracellular Ca2+ due to abnormalities in the Ca2+0-sensing receptor. In: Jameson L, ed. Contemporary endocrinology: hormone resistance syndromes. Totowa, NJ: Humana Press; 87–110
  5. Brown EM 1983 Four parameter model of the sigmoidal relationship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. J Clin Endocrinol Metab 56:572–581[Abstract]
  6. Gogusev J, Duchambon P, Hory B, Giovannini M, Goureau Y, Sarfati E, Drueke TB 1997 Depressed expression of calcium receptor in parathyroid gland tissue of patients with hyperparathyroidism. Kidney Int 51:328–336[Medline]
  7. Kifor O, Moore FD, Wang P, Goldstein M, Vassilev P, Kifor I, Hebert SC, Brown EM 1996 Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 81:1598–1606[Abstract]
  8. Farnebo F, Enbergt U, Grimelius L, Backdahl M, Schalling M, Larsson C, Farnebo LO 1997 Tumor-specific decreased expression of calcium sensing receptor messenger ribonucleic acid in sporadic primary hyperparathyroidism. J Clin Endocrinol Metab 82:3481–3486[Abstract/Free Full Text]
  9. Corbetta S, Mantovani G, Lania A, Borgato S, Vincentini L, E. Beretta E, Faglia G, Di Blasio AM, Spada A 2000 Calcium-sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia. Clin Endocrinol 52:339–348[CrossRef][Medline]
  10. Wada M, Furuya Y, Sakiyama J-i, Kobayashi N, Miyata S, Ishii H, Nagano N 1997 The calcimimetic compound NPS R-568 supresses parathyroid cell proliferation in rats with renal insufficiency. J Clin Invest 100:2977–2983[Medline]
  11. Kremer R, Bolivar I, Goltzman D, Hendy GN 1989 Influence of calcium and 1, 25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 125:935–941[Abstract]
  12. LeBoff MS, Rennke HG, Brown EM 1983 Abnormal regulation of parathyroid cell secretion and proliferation in primary cultures of bovine parathyroid cells. Endocrinology 113:277–284[Abstract]
  13. Nygren P, Gylfe E, Larsson R, Johansson H, Juhlin C, Klareskoq L, Akerstrom G, Rastad J 1988 Modulation of the Ca2+-sensing function of parathyroid cells in vitro and in hyperparathyroidism. Biochim Biophys Acta 968:253–260[Medline]
  14. McNeil SE, Hobson SA, Nipper V, Rodland KD 1998 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 273:1114–1120[Abstract/Free Full Text]
  15. Kurzchalia TV, Parton RG 1999 Membrane microdomains and caveolae. Curr Opin Cell Biol 11:424–431[CrossRef][Medline]
  16. Okamoto T, Schlegel A, Scherer PE, Lisanti MP 1998 Caveolins, a family of scaffolding proteins for organizing "pre-assembled signaling complexes" at the plasma membrane. J Biol Chem 273:5419–5422[Free Full Text]
  17. Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, Marks CB, Macaluso F, Russel RG, Li M, Pestell RG, Di Vizio D, Hou Jr H, Kneitz B, Lagaud G, Christ GJ, Edelman W, Lisanti MP 2001 Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem 276:38121–38138[Abstract/Free Full Text]
  18. Mora R, Bonilha VL, Marmorstein A, Scherer PE, Brown D, Lisanti MP, Rodriguez-Boulan E 1999 Caveolin-2 localizes to the Golgi complex but redistributes to plasma membrane, caveolae, and rafts when co-expressed with caveolin-1. J Biol Chem 274:25708–25717[Abstract/Free Full Text]
  19. Anderson RGW 1998 The caveolae membrane system. Annu Rev Biochem 67:199–223[CrossRef][Medline]
  20. Matveev SV, Smart EJ 2002 Heterologous desensitization of EGF receptors and PDGF receptors by sequestration in caveolae. Am J Physiol (Lung Cell Mol Physiol) 282:C935–C946
  21. Scheiffele P, Verkade P, Fra AM, Virta H, Simons K, Ikonen E 1998 Caveolin-1 and -2 in the exocytic pathway of MDCK cells. J Cell Biol 140:795–806[Abstract/Free Full Text]
  22. Razani B, Rubin CS, Lisanti MP 1999 Regulation of cAMP-mediated signal transduction via interaction of caveolins with the catalytic subunit of protein kinase A. J Biol Chem 274:26353–26360[Abstract/Free Full Text]
  23. Rybin VO, Xu X, Lisanti MP, Steinberg SF 2000 Differential targeting of ß-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. J Biol Chem 275:41447–41457[Abstract/Free Full Text]
  24. Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA, Michel T 1996 Endothelial nitric oxid synthase targeting to caveolae. J Biol Chem 271:22810–22814[Abstract/Free Full Text]
  25. Isshiki M, Anderson RGW 1999 Calcium signal transduction from caveolae. Cell Calcium 26:201–208[CrossRef][Medline]
  26. Kifor O, Diaz R, Butters R, Kifor I, Brown EM 1998 The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells. J Biol Chem 273:21708–21713[Abstract/Free Full Text]
  27. Fujimoto T, Miyawaki A, Mikoshiba K 1995 Inositol 1, 4, 5-trisphosphate receptor-like protein in plasmalemmal caveolae is linked to actin filaments. J Cell Sci 108:7–15[Abstract]
  28. Isshiki M, Ying Y, Fujita T, Anderson RGW 2002 A molecular sensor detects signal transduction from caveolae in living cells. J Biol Chem 277:43389–43398[Abstract/Free Full Text]
  29. Isshiki M, Ando J, Korenaga R, Kogo H, Fujimoto T, Fujita T, Kamiya A 1998 Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges. Proc Natl Acad Sci USA 95:5009–5014[Abstract/Free Full Text]
  30. Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, Schedl A, Haller H, Kurzchalia TV 2001 Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293:2449–2452[Abstract/Free Full Text]
  31. Galbiati F, Volonte D, Engelman JA, Watanabe G, Burk R, Pestell RG, Lisanti MP 1998 Targeted down-regulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade. EMBO J 17:6633–6648[CrossRef][Medline]
  32. Park H, Go Y-M, John PLS, Maland MC, Lisanti MP, Abrahamson DR, Jo H 1998 Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase. J Biol Chem 273:32304–32311[Abstract/Free Full Text]
  33. Engelman JA, Chu C, Lin A, Jo H, Ikezu T, Okamoto T, Kohtz DS, Lisanti MP 1998 Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. FEBS 428:205–211[CrossRef][Medline]
  34. Park DS, Lee H, Frank PG, Razani B, Nguyen AV, Parlow AF, Russell RG, Hulit J, Pestell RG, Lisanti MP 2002 Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade. Mol Biol Cell 13:3416–3430[Abstract/Free Full Text]
  35. Cohen AW, Park DS, Woodman SE, Williams TM, Chandra C, Shirani J, Pereira De Souza A, Kitsis RN, Russell RG, Weiss LM, Tang B, Jelicks LA, Factor SM, Shtutin V, Tanowitz HB, Lisanti MP 2003 Caveolin-1-deficient mice develop cardiac hypertrophy and show hyper-activation of the p42/44 MAP kinase cascade in areas of interstitial fibrosis and isolated cardiac fibroblasts. Am J Physiol Cell Physiol 284:C457–C474
  36. Gutkind JS 1998 The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 273:1839–1842[Free Full Text]
  37. Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, Brown EM 2001 Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol 280:F291–F302
  38. Aplin AE, Stewart SA, Assoian RK, Juliano RL 2001 Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of ELK-1. J Cell Biol 153:273–281[Abstract/Free Full Text]
  39. Brunet A, Roux D, Lenormand P, Dowd S, Keyse S, Pouyssegur J 1999 Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 18:664–674[CrossRef][Medline]
  40. Mithal A, Kifor O, Kifor I, Vassilev P, Butters R, Krapcho K, Simin R, Fuller F, Hebert SC, Brown EM 1995 The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca2+ is associated with marked reduction in the expression of extracellular Ca2+-sensing receptor messenger ribonucleic acid and protein. Endocrinology 136:3087–3092[Abstract]
  41. Martin KJ, Gonzalez EA 2001 The evolution of assays for parathyroid hormone. Curr Opin Nephrol Hypertens 10:575–580[CrossRef][Medline]
  42. Brown AJ, Zhong M, Ritter C, Brown EM, Slatopolsky E 1995 Loss of calcium responsiveness in cultured bovine parathyroid cells is associated with decreased calcium receptor expression. Biochem Biophys Res Commun 212: 861–867
  43. Galbiati F, Volonte D, Liu J, Capozza F, Frank PG, Zhu L, Pestell RG, Lisanti MP 2001 Caveolin-1 expression negatively regulates cell cycle progression by inducing G0/G1 arrest via p53/p21WAF1/Cip1-dependent mechanism. Mol Biol Cell 12:2229–2244[Abstract/Free Full Text]
  44. Roussanne M, Gogusev CJ, Hory B, Duchambon P, Souberbielle JC, Nabarra B, Pierrat D, Sarfati E, Drueke T, Bourdeau A 1998 Persistence of Ca2+-sensing receptor expression in functionally active, long-term human parathyroid cell cultures. J Bone Miner Res 13:354–362[CrossRef][Medline]
  45. Engelman JA, Zhang X, Galbiati F, Volonte D, Sotgia F, Pestell RG, Minetti C, Scherer PE, Okamato T, Lisanti MP 1998 Protein complex ’98 molecular genetics of the caveolin gene family: implications for human cancers, diabetes, Alzheimer disease, and muscular dystrophy. Am J Hum Genet 63:1578–1587[CrossRef][Medline]
  46. Lee SW, Reimer CL, Oh P, Campbell DB, Schnitzer JE 1998 Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells. Oncogene 16:1391–1397[CrossRef][Medline]
  47. Corbetta S, Lania A, Filopanti L, Vicentini L, Ballare E, Spada A 2002 Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells. J Clin Endocrin Metab 87:2201–2205[Abstract/Free Full Text]
  48. Vasef MA, Brynes RK, Sturm M, Bromley C, Robinson RA 1999 Expression of cyclin D1 in parathyroid carcinomas, adenomas, and hyperplasias: a paraffin immunohistochemical study. Mod Pathol 12:412–416[Medline]
  49. Hsi ED, Zukerberg LR, Yang W-I, Arnold A 1996 Cyclin D1/PRAD1 expression in parathyroid adenomas: an immunohistochemical study. J Clin Endocrinol Metab 81:1736–1739[Abstract]
  50. Hulit J, BashT, Fu M, Galbiati F, Albanese C, Sage DR, Schlegel A, Zhurinsky J, Shtutman M, Ben-Ze’ev A, Lisanti MP, Pestell RG 2000 The cyclin D1 gene is transcriptionally repressed by caveolin-1. J Biol Chem 275:21203–21209[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. Gosens, G. Dueck, W. T. Gerthoffer, H. Unruh, J. Zaagsma, H. Meurs, and A. J. Halayko
p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle
Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1163 - L1172.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. J. Costa, M. Senou, F. Van Rode, J. Ruf, M. Capello, D. Dequanter, P. Lothaire, C. Dessy, J. E. Dumont, M.-C. Many, et al.
Reciprocal Negative Regulation between Thyrotropin/3',5'-Cyclic Adenosine Monophosphate-Mediated Proliferation and Caveolin-1 Expression in Human and Murine Thyrocytes
Mol. Endocrinol., April 1, 2007; 21(4): 921 - 932.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
C. A. Syme, L. Zhang, and A. Bisello
Caveolin-1 Regulates Cellular Trafficking and Function of the Glucagon-Like Peptide 1 Receptor
Mol. Endocrinol., December 1, 2006; 20(12): 3400 - 3411.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
R. A. Chen and W. G. Goodman
Role of the calcium-sensing receptor in parathyroid gland physiology
Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1005 - F1011.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow