Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

Differential Calcium Requirements For Growth of Mouse Skin Epithelial and Fibroblast Cells

Concentrations of extracellular Ca⁺⁺ optimum for growth of cell types of mesodermal origin have been reported to be up to 100-fold higher than concentrations optimal for epidermal or other epithelial lining cells. In order to examine Ca⁺⁺ requirements of epithelial v. fibroblastic cells derived from a common tissue source, prior to prolonged culture, freshly isolated mouse epidermal keratinocytes, hair follicle cells and dermal fibroblasts were plated at high density or at clonal density in medium ranging from 0.014 to 1.4 mM Ca⁺⁺. Epithelial skin cells grew best at Ca⁺⁺ levels below 0.1 mM while dermal fibroblasts grew best at a Ca⁺⁺ concentration of 1.4 mM. the epithelial cell types exhibited marked morphologic changes in response to Ca⁺⁺, while the fibroblasts did not. These results suggest that the variations in Ca⁺⁺ response between lining epithelium and mesenchymal cells resulted from inherent differences in these cell types, but a mechanism for such differential effects has not yet been defined.     

Chronic heat-shock treatment driven differentiation induces apoptosis in Leishmania donovani

The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitro differentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmania donovani due to chronic heat-shock treatment of 37 ^°C that also mediates the differentiation of promastigotes to amastigotes. This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formation of preapoptotic bodies and involvement of Ca⁺⁺ in the apoptotic process. The flowcytometric analysis shows an early and steep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergistic action of Ca⁺⁺ ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway. Incubation of the culture with Ca⁺⁺ chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerably showing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infective individuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential of the physiological temperature of 37 ^°C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be put to.     

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts

Primary cultures of bone cells and skin fibroblasts were examined for their Ca⁺⁺ content, intracellular distribution and Ca⁺⁺ fluxes. Kinetic analysis of ⁴⁵Ca⁺⁺ efflux curves indicated the presence of three exchangeable Ca⁺⁺ compartments which turned over at different rates: a “very fast turnover” (S₁), a “fast turnover” (S₂), and a “slow turnover” Ca⁺⁺ pool (S₃). S₁ was taken to represent extracellular membrane-bound Ca⁺⁺, S₂ represented cytosolic Ca⁺⁺, and S₃ was taken to represent Ca⁺⁺ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca⁺⁺ as compared with cultured fibroblasts. Most of this extra Ca⁺⁺ was localized in the “slow turnover” intracellular Ca⁺⁺ pool (S₃). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca⁺⁺: (1) In both types of cells serum caused an increase in the amount of Ca⁺⁺ in the “very fast turnover” Ca⁺⁺ pool, and an increase in the rate constant of ⁴⁵Ca⁺⁺ efflux from this pool, indicating a decrease in the strength of Ca⁺⁺ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca⁺⁺ in the “slow turnover” Ca⁺⁺ pool (S₃), an increase in the rates of Ca⁺⁺ efflux from the cells to the medium, and from S₃ to S₂, as well as a decrease in the rate of influx into S₃. (3) In bone cells the amount of Ca⁺⁺ in S₃ remained high in “serum activated” cells, the rate of efflux from S₃ to S₂ increased, and the rate of influx into S₃ also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca⁺⁺ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca⁺⁺ in bone cells and fibroblasts, in parallel with measurements of ³H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca⁺⁺ which was followed by a biphasic increase lasting until cell division.     

Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation

Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca⁺⁺ (Ca_i⁺⁺) and phospholipid degradation products regulate calpains in vitro, we measured Ca_i⁺⁺ and phospholipid degradation. Ca⁺⁺i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A₂ activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca⁺⁺i, 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia—an inhibition of phospholipid-mediated calpain activation. Interruption of phospholipase-mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury. © 1996 Wiley-Liss, Inc.     

l-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells

Oxidative stress and reactive oxygen species (ROS) generation can be influenced by G-protein coupled receptor (GPCR)-mediated regulation of intracellular Ca²⁺ ([Ca²⁺]_i) signaling. ROS production are much higher in proximal tubular (PT) cells; in addition, the lack of antioxidants enhances the vulnerability to oxidative damage. Despite such predispositions, PT cells show resiliency, and therefore must possess some inherent mechanism to protect from oxidative damage. While the mechanism in unknown, we tested the effect of l-ornithine, since it is abundantly present in PT luminal fluid and can activate Ca²⁺-sensing receptor (CaSR), a GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK2) cells, a PT cell line, and performed Ca²⁺ imaging and electrophysiological experiments to show that l-ornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that the operation of CaSR activated Ca²⁺ signaling in HK-2 cells mediated by the transient receptor potential canonical (TRPC) dependent receptor-operated Ca²⁺ entry (ROCE) using pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HyperRed indicator) to show that the l-ornithine-induced ROCE mediated [Ca²⁺]_i signaling protects from ROS production. Furthermore, we performed cell viability, necrosis and apoptosis assays, and mitochondrial oxidative gene expression to establish that presence of l-ornithine rescued the ROS-induced damage in HK-2 cells. Moreover, l-ornithine-activation of CaSR can reverse ROS production and apoptosis via mitogen-activated protein kinase p38 activation. Such nephroprotective role of l-ornithine can be useful as the translational option for reversing kidney diseases involving PT cell damage due to oxidative stress or crystal nephropathies.     

Junctional Membrane Uncoupling : Permeability transformations at a cell membrane junction

The permeability of the membrane surfaces where cells are in contact (junctional membranes) in Chironomus salivary glands depends on Ca⁺⁺ and Mg⁺⁺. When the concentration of these ions at the junctional membranes is raised sufficiently, these normally highly permeable membranes seal off; their permeability falls one to three orders, as they approach the nonjunctional membranes in conductance. This permeability transformation is achieved in three ways: (a) by iontophoresis of Ca⁺⁺ into the cell; (b) by entry of Ca⁺⁺ and/or Mg⁺⁺ from the extracellular fluid into the cell through leaks in the cell surface membrane (e.g., injury); or (c) by entry of these ions through leaks arising, probably primarily in the perijunctional insulation, due to trypsin digestion, anisotonicity, alkalinity, or chelation. Ca⁺⁺ and Mg⁺⁺ appear to have three roles in the junctional coupling processes: (a) in the permeability of the junctional membranes; (b) in the permeability of the perijunctional insulation; and (c) a role long known— in the mechanical stability of the cell junction. The two latter roles may well be closely interdependent, but the first is clearly independent of the others.     

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca⁺⁺ levels in other cell types in CF may represent a common primary dysfunction in Ca⁺⁺ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca⁺⁺ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca⁺⁺ (2.5×10^?3M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca⁺⁺ was not significantly different from that in BBMV not previously loaded with Ca⁺⁺. Furthermore, with TCA present in the incubation medium, Ca⁺⁺ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca⁺⁺. Also, Ca⁺⁺ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca⁺⁺ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.     

Microvillar Ca++ signaling: A new view of an old problem

Proceeding from the recent finding that the main components of the Ca⁺⁺ signal pathway are located in small membrane protrusions on the surface of differentiated cells, called microvilli, a novel concept of cellular Ca⁺⁺ signaling was developed. The main features of this concept can be summarized as follows: Microvilli are formed on the cell surface of differentiating or resting cells from exocytic membrane domains, growing out from the cell surface by elongation of an internal bundle of actin filaments. The microvillar tip membranes contain all functional important proteins synthesized such as ion channels and transporters for energy-providing substrates and structural components, which are, in rapidly growing undifferentiated cells, distributed over the whole cell surface by lateral diffusion. The microvillar shaft structure, a bundle of actin filaments, forms a dense cytoskeletal matrix tightly covered by the microvillar lipid membrane and represents an effective diffusion barrier separating the microvillar tip compartment (entrance compartment) from the cytoplasm. This diffusion barrier prevents the passage of low molecular components such as Ca⁺⁺ glucose and other relevant substrates from the entrance compartment into the cytoplasm. The effectiveness of the actin-based diffusion barrier is modulated by various signal pathways and effectors, most importantly, by the actin-depolymerizing/reorganizing activity of the phospholipase C (PLC)-coupled Ca⁺⁺ signaling. Moreover, the microvillar bundle of actin filaments plays a dual role in Ca⁺⁺ signaling. It combines the function of a diffusion barrier, preventing Ca⁺⁺ influx into the resting cell, with that of a high-affinity, ATP-dependent, and IP₃-sensitive Ca⁺⁺ store. Activation of Ca⁺⁺ signaling via PLC-coupled receptors simultaneously empties Ca⁺⁺ stores and activates the influx of external Ca⁺⁺. The presented concept of Ca⁺⁺ signaling is compatible with all established data on Ca⁺⁺ signaling. Properties of Ca⁺⁺ signaling, that could not be reconciled with the basic principles of the current hypothesis, are intrinsic properties of the new concept. Quantal Ca⁺⁺ release, Ca⁺⁺-induced Ca⁺⁺ release (CICR), the coupling phenomen between the filling state of the Ca⁺⁺ store and the activity of the Ca⁺⁺ influx pathway, as well as the various yet unexplained complex kinetics of Ca⁺⁺ uptake and release can be explained on a common mechanistic basis. J. Cell. Physiol. 180:19–34, 1999. © 1999 Wiley-Liss, Inc.     

Protoplasmic Swelling as a Symptom of Freezing Injury in Onion Bulb Cells : Its Simulation in Extracellular KCl and Prevention by Calcium

Freezing injury, in onion bulb tissue, is known to cause enhanced K⁺ efflux accompanied by a small but significant loss of Ca²⁺ following incipient freezing injury and swelling of protoplasm during the postthaw secondary injury. The protoplasmic swelling of the cell is thought to be caused by the passive influx of extracellular K⁺ into the cell followed by water uptake. Using outer epidermal layer of unfrozen onion bulb scales (Allium cepa L. cv Big Red), we were able to stimulate the irreversible freezing injury symptoms, by bathing epidermal cells in 50 millimolar KCl. These symptoms were prevented by adding 20 millimolar CaCl₂ to the extracellular KCl solution. Our results provide evidence that loss of cellular Ca²⁺ plays an important role in the initiation and the progression of freezing injury.     

Studies on the Active Transport of Calcium in Human Red Cells

The Ca⁺⁺ transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca⁺⁺ from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of ⁴⁵Ca infused inside red cells indicated that over 95% of all Ca⁺⁺ in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP¹, CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca⁺⁺ transport does not appear to involve exchange of Ca⁺⁺ with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca⁺⁺ transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca⁺⁺ transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.     

Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector

Summary We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34°C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca⁺⁺-resistant cells did not proliferate at the nonpermissive temperature (40°C), indicating that they depended on T antigen for their proliferation. The temperaturesensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40°C. The serum/Ca⁺⁺-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca⁺⁺-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.     

Different mechanism of LPS-induced calcium increase in human lung epithelial cell and microvascular endothelial cell: a cell culture study in a model for ARDS

Acute respiratory distress syndrome (ARDS) is a contemporary term incorporating the historic ‘acute lung injury’ and the colloquial term ‘shock lung’. ARDS remains a serious and enigmatic human disease, causing significant mortality. The mechanisms involved at the alveolar cell/capillary endothelial interface have been explored but to date we lack clarity on the role of intracellular calcium ([Ca²⁺]i) fluxes across this interface. To explore the mechanisms of Ca²⁺ induced inflammatory reaction in epithelial cells and pulmonary microvascular endothelial cells (HMVEC) located at the two sides of blood-air barrier, lung epithelial A549 and HMVEC cells were treated with LPS. Our results demonstrated that LPS evoked the increase of [Ca²⁺]i, TNF-α and IL-8 in both cells types. The [Ca²⁺]i increases involved intracellular but not extracellular Ca²⁺ sources in A549, but both intracellular and extracellular Ca²⁺ sources in HMVEC cells. The effects of LPS on both cells types were completely inhibited by the combination of LPS and CaSR-targeted siRNA. Furthermore, LPS-inhibited cell proliferations were significantly reversed by the combined treatment. Therefore, LPS induced different mechanisms of [Ca²⁺]i increase during the activation of CaSR in A549 and HMVEC cells, which translates into functional outputs related to ARDS.     

External Ca2+ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel whose dysfunction is associated with the onset of autosomal dominant polycystic kidney disease (ADPKD). PC2 contributes to Ca²⁺ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to the external Ca²⁺ regulation of PC2 channel function. In this study, we explored the effect of external Ca²⁺ on endogenous PC2 in wild type LLC-PK1 renal epithelial cells. We obtained whole cell currents at different external Ca²⁺ concentrations, and observed that the basal whole cell conductance in normal Ca²⁺(1.2 mM), decreased by 30.2% in zero (nominal) Ca²⁺ and conversely, increased by 38% in high external Ca²⁺(6.2 mM). The high Ca²⁺-increased whole cell currents were completely inhibited by either PC2 gene silencing, or intracellular dialysis with active, but not denatured by boiling, PC2 antibody. Exposure of cells to high Ca²⁺ was also associated with relocation of PC2 to the plasma membrane. To explore whether a Ca²⁺ sensing receptor (CaSR) was implicated in the external Ca²⁺ modulation of PC2 currents, we tested the effect of the CaSR agonists, spermine and the calcimimetic R-568, which largely mimicked the effect of high Ca²⁺ under Ca²⁺-free conditions. The CaSR agonist gentamicin also increased the PC2 currents in the presence of normal Ca²⁺. The presence of CaSR was confirmed by immunocytochemistry, which partially colocalized with the intracellular PC2 protein, in an external Ca²⁺-dependent manner. The data support a novel Ca²⁺ sensing mechanism for PC2 expression and functional regulation in renal epithelial cells.     

Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells

Background

The present study investigates the effects of high external calcium concentration ([Ca²⁺]_o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.

Methodology/Principal Findings

A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca²⁺]_o (0.37 mM); 2) high [Ca²⁺]_o (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca²⁺]_o and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca²⁺]_o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca²⁺]_o was not effective in this cell line. In small cells, both higher [Ca²⁺]_o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca²⁺]_o and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.

Conclusions/Significance

In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.     

Cellular responses to TGFβ and TGFβ receptor expression in human colonic epithelial cells require CaSR expression and function

CaSR and TGFβ are robust promoters of differentiation in the colonic epithelium. Loss of cellular responses to TGFβ or loss of CaSR expression is tightly linked to malignant progression. Human colonic epithelial CBS cells, originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer a unique opportunity in determining the functional linkage (if any) between CaSR and TGFβ. Knocking down CaSR expression abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca²⁺ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca²⁺ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR; there was no up-regulation of TGFβ receptor. It is concluded that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function.