Calcium insensitivity of FA-6, a cell line derived from a pancreatic cancer associated with humoral hypercalcemia, is mediated by the significantly reduced expression of the Calcium Sensitive Receptor transduction component p38 MAPK
© Morgan et al; licensee BioMed Central Ltd. 2006
Received: 06 September 2006
Accepted: 01 November 2006
Published: 01 November 2006
The Calcium-Sensing Receptor is a key component of Calcium/Parathyroid hormone homeostatic system that helps maintain appropriate plasma Ca2+ concentrations. It also has a number of non-homeostatic functions, including cell cycle regulation through the p38 MAPK pathway, and recent studies have indicated that it is required for Ca2+ mediated growth arrest in pancreatic carcinoma cells. Some pancreatic cancers produce pathogenic amounts of parathyroid like hormones, however, which significantly increase Ca2+ plasma concentrations and might be expected to block further cell growth. In this study we have investigated the expression and function of the p38 MAPK signaling pathway in Ca2+ sensitive (T3M-4) and insensitive (FA6) pancreatic cancer cell lines. FA-6 cells, which are derived from a pancreatic adenocarcinoma that secretes a parathyroid hormone related peptide, exhibit only very low levels of p38 MAPK expression, relative to T3M-4 cells. Transfecting FA-6 cells with a p38 MAPK expression construct greatly increases their sensitivity to Ca2+. Furthermore, the reduction of p38 MAPK in T3M-4 cells significantly reduces the extent to which high levels of Ca2+ inhibit proliferation. These results suggest that the low levels of p38 MAPK expression in FA-6 cells may serve to reduce their sensitivity to high concentrations of external Ca2+ that would otherwise block proliferation.
The Calcium -Sensing Receptor (CaR) was cloned a decade ago, and has proven to be an important component in Ca2+ homeostasis through its regulation of parathyroid hormone (PTH ). It also has a number of non-homeostatic functions including the control of ion channels and hormone secretion, and also in the regulation of cell cycle events . Most notably, Ca2+ stimulation of CaR promotes the proliferation of osteoblasts  and fibroblasts , together with a number of malignant cell types including myeloma and ovarian surface cancer , probably through activation of the EGF receptor . Conversely, the growth of some cells is blocked, including colonic  and some pancreatic carcinomas . In each case signaling through CaR is mediated by a specific Mitogen activated protein kinase (MAPK) pathway, characterized by and dependant upon p38 MAPK.
Hypercalcaemia due to the degradation of bone matrix by osteoclasts is commonly associated with a number of malignancies, most notably those of the breast , lung , testis and kidney , although it is also a feature of some pancreatic cancers . The mechanism frequently underlying this process is the secretion of PTH-related peptide (PTHrP) by cancer cells . This activates osteoblast cells that in turn promote osteoclasts to degrade the bone matrix, releasing large amounts of Ca2+. High Ca2+concentrations then stimulate further PTHrP secretion, resulting in increasingly severe bone loss and hypercalcaemia, and in many cases promotes further tumour growth [13, 14].
In view of the generalized anti-proliferative effect of Ca2+ on pancreatic adenocarcinoma cells (PACs), it is surprising that some PACs actually secrete high levels of PTHrP, thereby promoting hypercalcaemia. Here we compare two PAC derived cell lines, FA-6  and T3M-4 , which have similar characteristic except that FA-6 secretes high levels of PTHrP . Our results indicate that the relative insensitivity of FA-6 cells to Ca2+ is mediated by a constitutively low level of p38 MAPK expression in these cells.
Materials and methods
Cell culture and treatment
FA-6 and T3M-4 cells were maintained in RPMI 1640 supplemented with 10% FBS, 1% P/S and 1% B16 granulocyte-macrophage colony stimulating factor (GM-CSF) conditioned media, and incubated at 37°C, 5% CO2.
P38 MAPK transfection
The full length reading frame of p38 MAPK (NM_002745) was amplified by PCR and cloned into the pCMV-script vector (Stratagene, USA) to give pCMVp38+. As a control, we also cloned the p38 MAPK reading frame lacking the ATG translation start site (pCMVp38). Transfections for both FA-6 and T3M-4 were performed in 35 mm plates with cells at 80% confluency. 2 μg of each vector were used per plate, together with 6 μl of GeneJammer transfection reagent (Stratagene, USA), with other conditions as described in the manufacturer's protocol.
RNA extraction and RT-QPCR
Total RNA was extracted from FA-6 or T3M-4 cells using an RNeasy mini kit (Qiagen). 1 μg of RNA was used in subsequent reverse transcription reactions. This was mixed with a poly T15 oligo to 5 μg/ml and heated to 75°C for 5 minutes. After cooling on ice, the following additional reagents were added; dNTPs to 0.4 mM, RNase OUT (Promega) to 1.6 u/μl, Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLRvT) RnaseH- point mutant (Promega) to 8 u/μl and the appropriate buffer (supplied by the manufacturer) to x1 concentration. The mixture was incubated for one hour at 37°C, heated to 70°C for two minutes and cooled on ice.
QPCR reactions were all performed in a total volume of 50 μl. For each we used 1 μl of the M-MLRvT reaction (as described above), 0.2 nmols of each primer and 25 μl of pre-mixed QPCR components (Sigma). All reactions were cycled at 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 60 seconds, for 45 cycles.
QPCR was performed using the SYBR green labeling kit from Sigma. Thermal cycling and fluorescence detection was by a MX4000 (Stratagene Inc., USA). Semi-quantitative data was obtained by using measurements three cycles after reactions had risen above the base line, and were clearly in exponential increase. β-actin was used as a control for RNA recovery and cDNA synthesis, and all values are presented as a ratio of target to β-actin signal.
Detection of p38 MAPK protein by western blotting
Whole cell lystaes were prepared from FA-6 and T3M-4 cells and 15 μg was used for each detection. Blotting was performed using the Western Breeze Chemuliminescent kit (anti-rabbit, Invitrogen, UK), according to the manufactures instructions. Primary detection was by a rabbit anti-p38 MAPK antibody (ab7952, AbCam, UK), at a final concentration of 0.1 μg/ml. Blots were stripped and re-probed with rabbit anti-beta actin (Abcam, UK, ab8227) to act as a loading control.
siRNA silencing of p38 MAPK in T3M-4 cells
Cultured T3M-4 cells were plated in triplicate the day before the transfection procedure was started. Pre-validated siRNA (Ambion, id # 1634) was transfected at a final concentration of 100 nM. RNA was extracted from the cells 48 hours after transfection. mRNA levels were measured by real-time RT-PCR, as described above. As a positive control, we also transfected with siRNA to target GAPDH, and with a non-targeting siRNA, to act as a negative control. Both of these siRNAs are supplied in the Ambion 'siRNA starter kit', which was used for all of the transfections, using the manufacturer's protocol.
Results and discussion
In order to compare the expression of CaR and its downstream signaling components in PTHrP-expressing and non-expressing PAC lines, we selected FA-6  and T3M-4 . The former was established from a biopsied lymph node removed from a patient with PAC associated with humoral hypercalcaemia of malignancy, and secretes both PTHrP together with a TNFα-type bone reabsorbing activity. Likewise, T3M-4 was also established from a biopsied lymph node taken from a PAC patient, but unlike FA-6, T3M-4 is not known to secrete a bone-reabsorbing activity.
The analysis of differential transcription between cells and tissues is becoming increasingly important in diagnosis and prognosis, and also in understanding the physiology of different cell types. Studies using micro array based approaches have indicated that changes in the transcription of relatively small groups of genes may underlie the difference between normal and malignant states , and that these genes can be subdivide into functional distinct subclasses, for example those involved in proliferation and growth. In this study, we show that a single, stable transcriptional change between different pancreatic carcinoma cell lines may influence their response to distinct environmental signals, in this case the extracellular concentration of Ca2+. This change is in the transcription of p38 MAPK, a key component of the CaR-mediated proliferative/anti-proliferative affect of Ca2+ . It is intriguing that p38 MAPK should be so strongly downregulated in FA-6 cells, which are derived from a tumour that both promotes hypercalcaemia through PTHrP secretion whilst escaping its anti-proliferative effects seen in other PACs. Greatly reduced p38 MAPK expression is thus an example of a specific change in the CaR signaling pathway that can block its activity, and of a selection for a specific transcriptomic change that allows an escape from anti-proliferative signals.
- Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC: Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature. 1993, 366 (6455): 575-580. 10.1038/366575a0View ArticlePubMed
- Tfelt-Hansen J, Schwarz P, Brown EM, Chattopadhyay N: The calcium-sensing receptor in human disease. Front Biosci. 2003, 8: 377-390.View Article
- Yamaguchi T, Chattopadhyay N, Kifor O, Brown EM: Extracellular calcium (Ca2+o)-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+o on the function of ST2 cells. Endocrinology. 1998, 139 (8): 3561-3568. 10.1210/en.139.8.3561PubMed
- McNeil SE, Hobson SA, Nipper V, Rodland KD: Functional calcium-sensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium. J Biol Chem. 1998, 273 (2): 1114-1120. 10.1074/jbc.273.2.1114View ArticlePubMed
- Yamaguchi T, Yamauchi M, Sugimoto T, Chauhan D, Anderson KC, Brown EM, Chihara K: The extracellular calcium Ca2+o-sensing receptor is expressed in myeloma cells and modulates cell proliferation. Biochem Biophys Res Commun. 2002, 299 (4): 532-538. 10.1016/S0006-291X(02)02690-6View ArticlePubMed
- Tfelt-Hansen J, Yano S, John Macleod R, Smajilovic S, Chattopadhyay N, Brown EM: High calcium activates the EGF receptor potentially through the calcium-sensing receptor in Leydig cancer cells. Growth Factors. 2005, 23 (2): 117-123. 10.1080/08977190500126272View ArticlePubMed
- Kallay E, Kifor O, Chattopadhyay N, Brown EM, Bischof MG, Peterlik M, Cross HS: Calcium-dependent c-myc proto-oncogene expression and proliferation of Caco-2 cells: a role for a luminal extracellular calcium-sensing receptor. Biochem Biophys Res Commun. 1997, 232 (1): 80-83. 10.1006/bbrc.1997.6225View ArticlePubMed
- Racz GZ, Kittel A, Riccardi D, Case RM, Elliott AC, Varga G: Extracellular calcium sensing receptor in human pancreatic cells. Gut. 2002, 51 (5): 705-711. 10.1136/gut.51.5.705PubMed CentralView ArticlePubMed
- Grill V, Ho P, Body JJ, Johanson N, Lee SC, Kukreja SC, Moseley JM, Martin TJ: Parathyroid hormone-related protein: elevated levels in both humoral hypercalcemia of malignancy and hypercalcemia complicating metastatic breast cancer. J Clin Endocrinol Metab. 1991, 73 (6): 1309-1315.View ArticlePubMed
- Moseley JM, Kubota M, Diefenbach JH, Wettenhall RE, Kemp BE, Suva LJ, Rodda CP, Ebeling PR, Hudson PJ, Zajac JD: Parathyroid hormone-related protein purified from a human lung cancer cell line. Proc Natl Acad Sci USA. 1987, 84 (14): 5048-5052. 10.1073/pnas.84.14.5048PubMed CentralView ArticlePubMed
- Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Insogna KL, Weir EC, Broadus AE, Stewart AF: Identification of a novel 17, 000-dalton parathyroid hormone-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem. 1987, 262 (15): 7151-7156.PubMed
- Nagata N, Akatsu T, Kugai N, Yasutomo Y, Kinoshita T, Kosano H, Shimauchi T, Takatani O, Ueyama Y: The tumor cells (FA-6) established from a pancreatic cancer associated with humoral hypercalcemia of malignancy: a simultaneous production of parathyroid hormone-like activity and transforming growth factor activity. Endocrinol Jpn. 1989, 36 (1): 75-85.View ArticlePubMed
- Guise TA, Mundy GR: Cancer and bone. Endocr Rev. 1998, 19 (1): 18-54. 10.1210/er.19.1.18PubMed
- Tfelt-Hansen J, MacLeod RJ, Chattopadhyay N, Yano S, Quinn S, Ren X, Terwilliger EF, Schwarz P, Brown EM: Calcium-sensing receptor stimulates PTHrP secretion by PKC-, SEK1-, p38 MAPK- and ERK1/2-dependent pathways in H-500 cells. Am J Physiol Endocrinol Metab. 2003, 285 (2): E329-E337.View ArticlePubMed
- Okabe T, Yamaguchi N, Ohsawa N: Establishment and characterization of a carcinoembryonic antigen (CEA)-producing cell line from a human carcinoma of the exocrine pancreas. Cancer. 1983, 51 (4): 662-668. 10.1002/1097-0142(19830215)51:4<662::AID-CNCR2820510419>3.0.CO;2-XView ArticlePubMed
- Ashwell JD: The many paths to p38 mitogen-activated protein kinase activation in the immune system. Nat Rev Immunol. 2006, 6 (7): 532-540. 10.1038/nri1865View ArticlePubMed
- Stremmel C, Wein A, Hohenberger W, Reingruber B: DNA microarrays: a new diagnostic tool and its implications in colorectal cancer. Int J Colorectal Dis. 2002, 17 (3): 131-136. 10.1007/s00384-001-0370-7View ArticlePubMed
- Tfelt-Hansen J, Chattopadhyay N, Yano S, Kanuparthi D, Rooney P, Schwarz P, Brown EM: Calcium-sensing receptor induces proliferation through p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase but not extracellularly regulated kinase in a model of humoral hypercalcemia of malignancy. Endocrinology. 2004, 145 (3): 1211-1217. 10.1210/en.2003-0749View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.