Chondrocyte calcium-sensing receptor expression is up-regulated in early guinea pig knee osteoarthritis and modulates PTHrP, MMP-13, and TIMP-3 expression.

OBJECTIVE: Growth plate chondrocytes up-regulate calcium-sensing receptor (CaR) expression as they mature to hypertrophy. In cells other than chondrocytes, extracellular calcium-sensing via the CaR functions partly to promote expression of parathyroid hormone-related protein (PTHrP), a critical regulator of endochondral development. Moreover, PTHrP is up-regulated in human osteoarthritis (OA) and surgically induced rabbit OA cartilages and may promote both chondrocyte proliferation and osteophyte formation therein. Hence, we examined chondrocyte CaR-mediated calcium-sensing in OA pathogenesis. METHODS: We studied spontaneous knee OA in male Hartley guinea pigs. We also evaluated cultured bovine knee chondrocytes and immortalized human articular chondrocytes (CH-8 cells), employing the CaR calcimimetic agonist NPS R-467 or altering physiologic extracellular calcium (1.8 mM). RESULTS: Immunohistochemistry revealed that CaR expression became up-regulated in the superficial zone at 4 months of age in the guinea pig medial tibial plateau cartilage as early OA developed. CaR expression later became up-regulated in the middle zone. PTHrP content, measured by immunoassay, was significantly increased in the medial tibial plateau cartilage as OA developed and progressed. In cultured chondrocytic cells, CaR-mediated extracellular calcium-sensing, stimulated by the calcimimetic NPS R-467, induced PTHrP and matrix metalloproteinase (MMP)-13 expression and suppressed expression of tissue inhibitor of metalloproteinase (TIMP)-3 dose-dependently, effects shared by elevated extracellular calcium (3 mM). Extracellular calcium-sensing appeared essential for PTHrP and interleukin (IL)-1 to induce MMP-13 and for PTHrP 1-34 to suppress TIMP-3 expression. CONCLUSIONS: Chondrocyte CaR expression becomes up-regulated early in the course of spontaneous guinea pig knee OA. Chondrocyte CaR-mediated extracellular calcium-sensing promotes PTHrP expression, modulates the effects of PTHrP and IL-1, and promotes MMP-13 expression and TIMP-3 depletion. Our results implicate up-regulated extracellular calcium-sensing via the CaR as a novel mediator of OA progression.

Coherent expression chromosome cluster analysis reveals differential regulatory functions of amino-terminal and distal parathyroid hormone-related protein domains in prostate carcinoma.

Parathyroid hormone-related protein (PTHrP) has a number of cancer-related actions. While best known for causing hypercalcemia of malignancy, it also has effects on cancer cell growth, apoptosis, and angiogenesis. Studying the actions of PTHrP in human cancer is complicated because there are three isoforms and many derived peptides. Several peptides are biologically active at known or presumed cell surface receptors; in addition, the PTHrP-derived molecules can exert effects at the cell nucleus. To address this complexity, we studied gene expression in a DU 145 prostate cancer cell line that was stably transfected with control vector, PTHrP 1-173 and PTHrP 33-173. With this model, regulatory effects of the amino-terminal portion of PTHrP would result only from transduction with the full-length molecule, while effects pertaining to distal sequences would be evident with either construct. Analysis of the expression profiles by microarrays demonstrated nonoverlapping groups of differentially expressed genes. Amino-terminal PTHrP affected groups of genes involved in apoptosis, prostaglandin and sex steroid metabolism, cell-matrix interactions, and cell differentiation, while PTHrP 33-173 caused substantial increases in MHC class I antigen expression. This work demonstrates the distinct biological actions of the amino-terminus compared to distal mid-molecule or carboxy-terminal sequences of PTHrP in prostate carcinoma cells and provides targets for further study of the malignant process.

Monitoring of skeletal progression of prostate cancer by GFP imaging, X-ray, and serum OPG and PTHrP.

BACKGROUND: Prostate cancers (PCas) produce factors that can serve as biomarkers for tumor metastasis and bone progression. Transduced GFP expression by cancer cells can be imaged to monitor therapy. We exploited both concepts by developing a GFP-expressing PCa cell line that expresses PTHrP and studying it in an animal model of malignancy with methods that assess the skeletal progression of this tumor. METHODS: We developed a GFP-producing PCa cell line by stable transduction of PC-3 PCa cells. This PC-3 variant was used to study tumor progression in an immunocompromised mouse model. Skeletal progression of the PCa cells and the effects of pamidronate administration were evaluated radiologically, fluorometrically, and by measurement of serum tumor markers. RESULTS: The PC-3 cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3 cells could be monitored by GFP optical imaging, X-ray, and by measurements of tumor products in serum, notably PTHrP and OPG. Pamidronate treatment reduced tumor burden as assessed at autopsy by imaging and biomarkers. CONCLUSIONS: Pamidronate treatment exhibited anti-tumor effects that were reflected by decreases in serum PTHrP, OPG, and by GFP and radiological imaging procedures. Imaging of GFP expression enables real-time monitoring of tumor growth in the bone. PTHrP and OPG may be useful as tumor biomarkers for PCa that has metastasized to bone. This novel human PCa model can be used to study the clinical potential of diagnostic and therapeutic modalities in the skeletal progression of PCas.

Parathyroid hormone-related protein regulates the growth of orthotopic human lung tumors in athymic mice.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) has growth regulatory effects for many malignant cells and may influence the progression of carcinomas of the breast, prostate, and lung. In the current study, the authors investigated the in vivo and in vitro effects of PTHrP neutralizing antibody and PTHrP treatment on the growth of BEN cells, a human lung squamous cell carcinoma line that expresses PTHrP and its receptor. METHODS: Orthotopic lung tumors were produced in 20 athymic mice with BEN-GFP cells (a clonal line that stably expresses green fluorescent protein [GFP]) by instilling suspensions of 3 x 10(6) cells per mouse into the lungs of anesthetized animals. The mice were divided into 2 groups receiving either subcutaneous mouse antihuman PTHrP antibodies or irrelevant mouse immunoglobulin (Ig) G (150 microg) twice weekly. RESULTS: After 30 days, 6 of 10 mice receiving anti-PTHrP antibodies had lung tumors visible on macroscopic inspection, but only 1 of the 10 mice treated with irrelevant IgG had a lung tumor that was of that size (P < 0.01). GFP fluorescence was significantly greater in lung homogenates of the PTHrP antibody-treated mice than in the mice treated with IgG (6006 +/- 411 vs. 2907 +/- 282 relative fluorescent units, respectively; P < 0.001). Although neutralizing antibodies stimulated BEN cell lung tumor growth, exogenous PTHrP 1-34 treatment (0.01-1 nM) inhibited the growth of cultured BEN cells by approximately 40%. CONCLUSIONS: Although PTHrP expression has been reported to be associated with more aggressive malignancies, the data from the current study suggest that PTHrP 1-34 was a paracrine growth inhibitor in BEN human lung carcinoma cells. The growth-related effects of PTHrP are complex, and can be both stimulatory and inhibitory.

Human pancreatic adenocarcinomas express parathyroid hormone-related protein.

PTH-related protein (PTHrP) is expressed in many common malignancies such as breast and prostate cancer and can regulate their growth. Little is known, however, about the role of PTHrP in pancreatic adenocarcinoma. To study PTHrP in pancreatic exocrine cancer, we studied its expression in pancreatic cancer cell lines and surgical specimens. Eight human pancreatic adenocarcinoma cell lines were evaluated: AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, PANC-1, PANC-28, and PANC-48. Murine monoclonal antibodies to the amino-terminal (1-34), mid-region (38-64), and carboxyl-terminal peptides (109-141) of PTHrP were used to identify cellular PTHrP and secreted PTHrP, including Western blotting and immunocytochemical staining for PTHrP from each cell line. Cellular PTHrP was detected in all cell line extracts by both Western blotting and immunoassay. CFPAC-1, derived from a pancreatic liver metastasis, had the highest concentration of PTHrP, and MIA PaCa-2, derived from primary pancreatic adenocarcinoma, had the lowest. PTHrP was localized by immunocytochemical staining in the cytoplasm in all but one cell line, and both nuclear and cytoplasmic immunostaining were observed in the MIA PaCa-2 and PANC-1 cells. Secretion of PTHrP into cell medium was also observed for each cell line and paralleled intracellular PTHrP levels. Evidence for differential processing of PTHrP expression was provided by studies demonstrating different patterns of PTHrP among the cell lines when assessed by PTHrP immunoassays directed against different PTHrP peptides. In specific, PTHrP secretion measured by a PTHrP-(38-64) assay was highest for BxPC-3, whereas the highest levels of secreted PTHrP-(109-141) occurred in CFPAC-1 and PANC-1. Growth of AsPC-1 cells was stimulated in a dose-dependent manner by PTHrP-(1-34). Immunostaining from archival tissue of patients with pancreatic adenocarcinoma revealed strong PTHrP expression in all 14 specimens. All patients were eucalcemic preoperatively. These results demonstrate that PTHrP is commonly expressed in pancreatic cancer. Our data suggest that PTHrP may have growth-regulating properties in pancreatic adenocarcinoma cells, but further studies are required.

Parathyroid hormone-related protein induces interleukin 8 production by prostate cancer cells via a novel intracrine mechanism not mediated by its classical nuclear localization sequence.

PTHrP (parathyroid hormone-related protein) overexpression by prostate carcinoma cells has been implicated in tumor progression. Although the biological effects of PTHrP can be mediated by the G-protein-coupled PTH/PTHrP receptor, PTHrP also has intracrine actions mediated by a nuclear localization sequence at residues 87-107. We investigated the effect of PTHrP transfection and treatment on production by prostate carcinoma cells of IL (interleukin)-8, which can regulate prostate cancer growth by angiogenic activity and growth-promoting effects. Six prostate cancer cell lines exhibited constitutive expression of PTHrP and IL-8 that were significantly correlated (r = 0.93; P < 0.01). We transfected wild-type and mutant PTHrP into these cells. Wild-type PTHrP1-173 and PTHrP33-173 lacking the PTH/PTHrP receptor-binding domain induced a 3-fold stimulation of IL-8 production but not production of another angiogenic factor, vascular endothelial growth factor. Transfection of the COOH-terminal truncation mutant PTHrP1-87 induced a 5-fold simulation of IL-8 and a 3-fold increase in IL-8 mRNA. Cells transfected with PTHrP1-87 and 1-173 also showed increased cell proliferation. In contrast, exogenous PTHrP1-34 and 1-86 peptides did not significantly affect IL-8 production; moreover, PTHrP-neutralizing antibodies did not inhibit the production of IL-8 by transfected PTHrP. Additional transfection studies with progressively COOH-terminally truncated PTHrP1-87 defined a 23-amino acid sequence, PTHrP65-87, required for PTHrP1-87 to robustly stimulate IL-8 in prostate cancer cells. Confocal microscopy and immunoassay demonstrated PTHrP1-87 nuclear localization. Our results demonstrate that PTHrP acts to induce IL-8 production in prostate cancer cells via an intracrine pathway independent of its classical nuclear localization sequence. This novel pathway could mediate the effects of PTHrP on the progression of prostate cancer.

The tetrabasic KKKK(147-150) motif determines intracrine regulatory effects of PthrP 1-173 on chondrocyte PPi metabolism and matrix synthesis.

Expression of PTHrP is a major regulator of growth cartilage development and also becomes robust in osteoarthritic cartilage. We further defined how PTHrP 1-173, which we observed to be the preferentially expressed PTHrP isoform in normal and osteoarthritic cartilage, functions in chondrocytes. We transfected both immortalized human juvenile costal chondrocytes (TC28 cells) and rabbit articular chondrocytes with wild-type PTHrP 1-173 and mutants of putative PTHrP 1-173 endoproteolytic processing sites. Wild-type PTHrP 1-173 inhibited collagen synthesis and decreased extracellular PPi (which critically regulates hydroxyapatite deposition) by 50-80% in both chondrocytic cell types. In contrast, PTHrP 1-173 mutated at the PTHrP 147-150 motif KKKK (but not the other site-directed mutants) and increased both extracellular PPi and collagen synthesis by >50%. Synthetic PTHrP 140-173 mutated at amino acids 147-150 and also increased extracellular PPi, and wild-type 140-173 decreased extracellular PPi in permeabilized cells. The 147-nuclear localization of PTHrP. We conclude that the tetrabasic 147-150 motif functions to determine how PTHrP 1-173 regulates collagen synthesis and levels of extracellular PPi by an intracrine mechanism in chondrocytes, and it may prove useful as a therapeutic target for regulation of mineralization.

Splenic, thymic, bony and lymph node metastases from orthotopic human lung carcinomas in immunocompromised mice.

In orthotopic animal models of human lung cancer, bone and lymph node metastases have been observed with high frequency after periods of a few weeks, but metastases to other organs are rare. This study evaluated development of distant metastases over a six-month period in a model of orthotopic lung carcinoma in immunocompromised mice. Human A549 lung adenocarcinoma cells were stably transfected to express high levels of green fluorescent protein. Suspensions of 1 x 10(6) cells were instilled into the lungs of athymic and SCID mice to produce orthotopic human lung carcinomas. All animals had primary tumors at termination of the experiment six months later. Splenic metastases and lymph node metastases were present in 70% of the animals and two of the three SCID mice had thymic metastases. Three animals had bony metastases. Thus, a high percentage of immunocompromised mice with orthotopic lung carcinomas ultimately develop metastases.

Parathyroid hormone-related protein reduces alveolar epithelial cell proliferation during lung injury in rats.

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells and could be a regulatory factor for alveolar epithelial cell proliferation after lung injury. We investigated lung PTHrP expression in rats exposed to 85% oxygen. Lung levels of PTHrP were significantly decreased between 4 and 8 days of hyperoxia, concurrent with increased expression of proliferating cell nuclear antigen and increased incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA in lung corner cells. PTHrP receptor was present in both normal and hyperoxic lung. To test whether the fall in PTHrP was related to cell proliferation, we instilled PTHrP into lungs on the fourth day of hyperoxia. Eight hours later, BrdU labeling in alveolar corner cells was 3.2 +/- 0.4 cells/high-power field in hyperoxic PBS-instilled rats compared with 0.5 +/- 0.3 cells/high-power field in PTHrP-instilled rats (P < 0. 01). Thus PTHrP expression changes in response to lung injury due to 85% oxygen and may regulate cell proliferation.

Parathyroid hormone-related protein in patients with primary breast cancer and eucalcemia.

Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in breast cancer and other malignancies. We studied circulating PTHrP levels with three different immunoassays directed against different parts of the PTHrP molecule in 48 patients with breast cancer and eucalcemia. The methods used were: (a) a RIA with antibodies directed toward the midregion (63-78); (b) an immunofluorometric assay with two antibodies against 1-34 and 38-67; and (c) an immunoradiometric assay with antibodies against 1-40 and 1-72. Although most patients had PTHrP levels indistinguishable from normal when measured by all three methods, four patients had increased serum levels in the IFMA. PTHrP was detected by immunohistochemistry in tumors from nearly all patients. One patient with elevated PTHrP in plasma measured by IFMA showed intense staining of tumor by immunohistochemistry; the tumor was histologically graded as III (severe) and was the largest of all tumors in this patient group. The IFMA can identify increased serum PTHrP in some patients with breast cancer who are not hypercalcemic. This assay may be especially useful in screening patients for this tumor during a relative early phase of the disease.

Hypercalcemia due to parathyroid hormone-related protein secretion by melanoma.

About 1-2% of melanoma patients develop hypercalcemia. We report hypercalcemia without bone metastasis in a 46-year-old woman with advanced melanoma. The hypercalcemia was associated with elevated serum parathyroid hormone-related protein (PTHrP) levels. An even higher concentration (10 times the serum level) in pleural effusion caused by pleural metastases implied that the source of the increased circulating PTHrP was the melanoma. Immunohistochemical staining of paraffin sections, performed using a monoclonal antibody (9H7) against the peptide sequence 109-141 of human PTHrP, detected PTHrP in the cytoplasm and nucleoli of melanoma cells in an autopsy specimen but not in specimens from this patient prior to onset of hypercalcemia. Considering the evidence, it is very likely that PTHrP production by melanoma caused hypercalcemia in this patient.

Parathyroid hormone-related protein, an autocrine growth inhibitor of alveolar type II cells.

Parathyroid hormone-related protein (PTHRP) is an autocrine regulator of differentiation for type II pneumocytes [R. H. Hastings, D. Summers-Torres, T. C. Cheung, L. S. Ditmer, D. W. Burton, E. M. Petrin, R. G. Spragg, J. Li, and L. J. Deftos. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L353-L361, 1996]. We investigated autocrine effects on growth by decreasing endogenous PTHRP in rat type II cells. Cultured cells were incubated with antibodies against PTHRP-(1-34) (8B12) or PTHRP-(109-141) (9H7) or an irrelevant antibody (1 microg/ml) for 3 days. Conditioned media from the irrelevant antibody group contained 143 +/- 8 fg PTHRP/ 100,000 cells. The 8B12 and 9H7 reduced levels to 45 +/- 8 and 88 +/- 16 fg PTHRP/100,000 cells, respectively (n = 4 cell isolations, P < 0.05). Cells treated with the PTHRP antibodies nearly tripled in number. The irrelevant antibody had no effect on growth. Exogenous PTHRP-(1-34) (2.5 nM) blocked the growth-stimulating effect of 9H7. Instilled intratracheal 8B12 and 9H7 induced expression of proliferating cell nuclear antigen in clusters of alveolar cells in rats. Clustered cells expressed surfactant apoproteins and cytokeratin 19. These data suggest that endogenous PTHRP-(1-34) inhibits proliferation of type II cells in vivo and in vitro.

Hypercalcemia and systemic lupus erythematosus.

Hypercalcemia is commonly caused by the increased production of parathyroid hormone-related protein (PTHrP) by a malignancy. In fact, the demonstration of increased PTHrP production in a patient with hypercalcemia is virtually pathognomonic of malignancy. We studied a patient with systemic lupus erythematosus (SLE), generalized lymphadenopathy, and hypercalcemia. Immunohistology of 2 biopsied lymph nodes revealed the abundant expression of PTHrP and the absence of malignant transformation. Although apparently rare, PTHrP production by non-malignant lymphoid tissue may occur in SLE and should be considered in the differential diagnosis of hypercalcemia.

Transcriptional regulation of the human chromogranin A gene by its 5' distal regulatory element: novel effects of orientation, structure, flanking sequences, and position on expression.

In previous studies of human chromogranin A (hCgA) gene expression, we had identified a 27 bp distal regulatory element (DRE) located between -576 and -550 bp that interacts with a CRE-containing promoter to enhance gene transcription specifically in neuroendocrine (NE) cells. To characterize the DRE we have now mutated its various parts and assessed the effects on protein binding by electrophoretic mobility shift assays (EMSAs) and hCgA transcription within BEN cells. We found that the sequence TGACTAA, an AP-1 binding site that we refer to as DRE-AP-1, was necessary but not sufficient to produce the DRE's enhancer effect. Moreover, while AP-1 (Jun/Fos) bound this site, binding was not correlated with transcriptional effects. Protein binding by the DRE-AP-1 could be attenuated by mutations of its flanking sequences, and transcriptional enhancement by the DRE was dependent on its orientation and spatial relationship to the hCgA proximal promoter. Mutation of the DRE-AP-1 to a consensus AP-1 did not produce greater transcriptional activity, even though it increased binding of nuclear factors. Co-transfection with c-jun and/or c-fos expression plasmids showed that the DRE was unresponsive to the over-expressed AP-1 proteins. Co-transfection with wild-type DRE oligonucleotides competitively inhibited DRE-mediated transcription, while co-transfection with mutant DRE oligonucleotides had a lesser effect. Our studies indicate that transcriptional enhancement of hCgA by the DRE is dependent on a unique NE-specific DRE-binding factor, which we refer to as DBF, that specifically and directionally binds the DRE to assemble and synergize a functional transcription complex.

Immunoassay and immunohistology studies of chromogranin A as a neuroendocrine marker in patients with carcinoma of the prostate.

OBJECTIVES: Neuroendocrine differentiation in carcinoma of the prostate is characterized by the expression of neuroendocrine cell products such as chromogranin A (CgA). We studied serum levels and tissue staining for CgA in prostate cancer to assess their clinical value. METHODS: In 82 patients with prostate cancer, serum specimens were obtained at diagnosis and studied by both CgA and prostate-specific antigen (PSA) immunoassays. In 43 additional patients with prostate cancer, paraffin-embedded tissue from core biopsies or transurethral resections and serum samples were studied, respectively, by immunohistology and immunoassay for CgA. RESULTS: In serum samples from the 82 patients in whom CgA and PSA levels were measured, 26 of 82 (32%) had an elevated CgA (greater than 200 ng/mL), and 36 of 82 (44%) had an elevated PSA (greater than 4.0 ng/mL). Of the patients with Stage D2 cancer, 11 of 18 (61%) had an elevated CgA and 6 of 18 (33%) had an elevated PSA. Four of 5 patients with local recurrence had an elevated CgA, but only 1 patient had an elevated PSA. Of the 43 patients in whom serum and tissue CgA studies were performed, 12 (28%) had elevated serum CgA, and 15 of the 43 (35%) had CgA staining in their prostate tissue. Of the 14 of these patients with D2 disease (distant metastases), 9 (64%) had elevated serum levels of CgA and 6 (43%) had positive staining in their prostate tissue. Of the 9 patients with Stage D2 disease and elevated serum CgA, 6 had a normal serum PSA. CONCLUSIONS: Our studies complement those of others and indicate that CgA has potential as a clinically useful serum and tumor marker for prostate cancer. Serum CgA measurements can identify some patients with advanced disease who do not have elevated serum PSA. However, further studies in larger groups of patients are needed to define the clinical value of CgA as a marker for prostate cancer.

Elimination of the carboxy-terminal sequences of parathyroid hormone-related protein 1-173 increases production and secretion of the truncated forms.

The endoproteolytic processing of polypeptides at basic residues into distinct biologically active peptides is a common theme in prohormone maturation and processing. PTH-related protein (PTHrP) 1-173 contains eight putative endoproteolytic consensus sites that include a mono-arginyl (R37), paired basic (RR154-155), and related basic residue motifs (RLKR-4 to -1, RRR19-21, KKKK88-91, KRK96-98, KKKRR102-106, and KKKK147-150). To analyze the primary structural determinants involved in the posttranslational processing and secretion of PTHrP 1-173, we constructed a series of nonsense mutants that code for carboxy-terminal truncated polypeptides. Since the basic residue motifs are probable sites of endoproteolysis, these sites and the residues downstream were serially eliminated, thereby creating PTHrP 1-152, 1-146, 1-101, 1-95, 1-87, 1-36, and 1-18. The wild type PTHrP 1-173 and nonsense mutant constructs were transiently transfected into two cell lines, COS-1 and SK-N-BE(2). The COS-1 cells have a constitutive secretory pathway, whereas the neuroblastoma-derived BE-2 cells have, in addition, a regulated secretory pathway. PTHrP was measured in the conditioned media and cell extracts of the transfected cells with two peptide-specific RIAs. In COS-1 cells, PTHrP truncation mutants 1-152, 1-146, 1-101, 1-95, and 1-87 were present relative to wild type isoform 1-173, at 4.4-, 3-, 19-, 12-, and 57-fold excess, respectively; a similar pattern was also detected with BE-2 transfected cells, although the relative increases above the quantities of PTHrP 1-173 were not as dramatic. As the carboxy-terminal sequences were eliminated, the amount of total and secreted PTHrP increased, and the percentage found within the cell decreased. In COS-1 cells, 10.5% of the total PTHrP 1-173 was intracellular, whereas only 1% of the total PTHrP 1-87 was intracellular. In BE-2 cells, 54% of the total PTHrP 1-173 and only 9% of the total 1-87 mutant were intracellular. In COS-1 cells, a time course analysis demonstrated that PTHrP 1-87 and 1-95 were detectable in media 3 h after transfection, whereas 1-173 was barely detectable after 24 h. Our studies suggest that the carboxy-terminal sequence of PTHrP 1-173 is responsible for the intracellular degradation of this polypeptide, which may be the endogenous cellular mechanism that regulates the amount of processed and secreted PTHrP.

The humoral hypercalcemia of benignancy. A newly appreciated syndrome.

It has become recently appreciated that the hypercalcemia of malignancy is commonly caused by the increased production of parathyroid hormone-related protein (PTHrP) by the cancer. In fact, the demonstration of increased PTHrP production in a patient with hypercalcemia is regarded as pathognomonic of malignancy. The authors describe a patient with a benign ovarian lesion that produced PTHrP and caused hypercalcemia. They identify other reports of hypercalcemia associated with hypercalcemia and benign tumors, and refer to this syndrome as the humoral hypercalcemia of benignancy. Although apparently rare, a benign PTHrP-producing tumor should be considered in the differential diagnosis of hypercalcemia.

Parathyroid hormone-related protein, an autocrine regulatory factor in alveolar epithelial cells.

Alveolar epithelial cells in vivo, primary cultures of adult rat type II cells, and human A549 alveolar carcinoma cells express parathyroid hormone-related protein (PTHrP). Here we demonstrated that type II cells and A549 cells also express the PTHrP receptor and that they exhibit differentiation-related responses to the amino-terminal PTHrP fragment, PTHrP-(1-34). PTHrP receptor expression in A549 cells was shown by detection of a 0.3-kb reverse transcriptase polymerase chain reaction product formed by primers specific for PTHrP receptor. In situ hybridization studies localized the site of production of PTHrP and PTHrP receptor mRNA in rat lung cells with morphology and location typical of type II cells. Primary cultures of such type II cells also expressed PTHrP receptor mRNA. Incubation with PTHrP-(1-34) stimulated disaturated phosphatidylcholine (DSPC) synthesis in A549 cells and increased the release of newly synthesized DSPC by cultured type II cells and A549 cells. In addition, PTHrP-(1-34) increased the number of lamellar bodies per type II cell and increased their expression of alkaline phosphatase in a dose-dependent manner. Thus PTHrP-(1-34) promoted a differentiated type II cell phenotype. Since cultured type II cells, alveolar epithelial cells in vivo, and A549 cells express PTHrP and the PTHrP receptor, PTHrP-(1-34) may be an autocrine regulatory factor in type II cells and lung cancer cells.

Identification and characterization of a neuroendocrine-specific 5'-regulatory region of the human chromogranin A gene.

Chromogranin A (CgA) expression is specific to cells of endocrine and neuroendocrine (NE) tissues. Our transfection studies with CgA have identified two DNA regions 5' of the transcription start site that regulate CgA gene transcription: a distal regulatory region (DRR) located between -726 and -455, and a proximal regulatory region (PRR) between -60 and -26. In studies of the DRR using four human NE and six human non-NE cell lines, we demonstrated enhanced transcription of DRR-containing CgA-GH plasmids by the NE cells as a group compared to the non-NE cells. DNase I footprinting identified a protected area in the DRR from -570 to -555 base pairs (bp) composed of the sequence TAATGATGACTAAACA. Centered in this sequence is the simian virus 40 version of the activator protein-1-binding site, TGACTAA. Electrophoretic mobility shift assays (EMSAs) with an oligonucleotide containing the 27 bp of the DRR between -576 and -550, which we refer to as the distal regulatory element (DRE), produced a specific complex with the NE BEN and non-NE COS-1 cell nuclear extracts. The addition of c-Jun and c-Fos antibodies produced strong supershifts of the complex generated by COS-1 extract, but very weak supershifts of the complex formed by BEN extract. These EMSA studies suggest that NE cells such as BEN contain unique nuclear factors distinguishable from activator protein-1 that interact with the DRE. The enhancer effect of the 271-bp DRR could be replaced by the 27-bp DRE in both CgA and calcitonin promoter constructs in BEN cells. Replacement of the DRR with the DRE resulted in a further increase in expression from these plasmids, suggesting the presence of suppressor sequences in the DRR. In transfection studies of the PRR, deletion of its cAMP response element (CRE) dramatically lowered transcription. In addition to demonstrating that its CRE can bind CRE-binding protein, EMSAs with the PRR demonstrated that an intervening sequence between the CRE and the TATA box formed a complex with BEN cell nuclear extract. Our studies demonstrate that both the PRR and DRR are important for high level transcription of the CgA gene in NE cells. The presence of both distal and proximal 5'-regulatory regions in the human CgA gene indicates a complex mechanism of transcriptional regulation. Although the PRR is important for the formation of a functional transcription complex at the TATA region, the DRR is important for the enhancement of CgA gene expression in NE cells.