Coexpression of PTHrP and PTH/PTHrP receptor in a myoepithelial cell line derived from normal human breast

Parathyroid hormone-related peptide (PTHrP) is the cause of humoral hipercalcaemia of malignancy syndrome (HHM). It is known that the peptide as well as its receptors are widely distributed in many normal organs and tissues, where it influences an array of diverse functions which are realized through paracrine or autocrine pathway. PTHrP is present in large amounts in lactating mammary gland but its function is not fully elucidated. In this study, production of parathyroid hormone-related peptide (PTHrP) by the Hs578Bst cell line corresponding to mammary myoepithelial cells was examined by immunocytochemistry. Using RNA extracted from these cells we analyzed expression of mRNA for PTHrP and for the PTH/PTHrP receptor by RT-PCR. The obtained results demonstrated that Hs578Bst cells produced PTHrP and synthesized mRNA for PTHrP and PTH/PTHrP type I receptor. It provides evidence that myoepithelial cells are target cells for PTHrP. The data support that PTHrP may be an important autocrine/paracrine factor, involved in the regulation of myoepithelial cell function as well as in growth and differentiation of the mammary gland.     

Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells

Type V collagen is a "minor" component of normal human breast stroma, which is subjected to over-deposition in cases of ductal infiltrating carcinoma (DIC). We reported that, if used as a culture substrate for the DIC cell line 8701-BC, it exhibited poorly-adhesive properties and restrained the proliferative and motile behavior of the cell subpopulation able to attach onto it. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of 8701-BC cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by type V collagen was of the apoptotic type by (i) microscopic detection and quantitation of Apoptag-labeled cells, (ii) analysis of the expression levels of selected genes coding for apoptosis-linked factors, caspases, and stress-response proteins by conventional and semi-quantitative multiplex PCR, and (iii) evaluation of the extent of caspase activation by chromogenic assay. We report here that type V collagen is able to determine an increase in the percentage of Apoptag-positive cells, to up-regulate Bcl-xS, Bad, Dap kinase, hsf-1, mthsp75, caspase-1, -5, -8, -9, and -14, whilst down-regulating Bcl-2, Bcl-xbeta, and hsp60. Treatment of cell lysates with chromogenic tetrapeptide substrates specific for caspase-1, -5, -8, and -9 demonstrated a marked increase of enzymatic activity in the presence of type V collagen. Our data validate 8701-BC cell line as a suitable "in vitro" model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on primary DIC cells.     

PTH-1R responses to PTHrP and regulation by vitamin D in keratinocytes and adjacent fibroblasts

Vitamin D and PTHrP are essential for the differentiation of keratinocytes and epidermal development. The action of PTHrP on skin is mediated via its PTH-1R receptors present in both epidermal and dermal cells. This suggests that PTHrP may have a paracrine/autocrine role, and its receptors may act in association or in negative cooperativity. We compared the intracellular signaling pathways in response to PTHrP (1-34) and to various PTHrP peptides, the N-terminal (1-34), Mid region (67-89), and C-terminal (107-139) fragments, and the possible modulation of PTHrP and its receptor mRNA expressions by vitamin D. Adjacent dermal fibroblasts as freshly isolated keratinocytes expressed both PTHrP and PTH-1R mRNAs, and responded to the various PTHrP fragments. bPTH and PTHrP(1-34) increased both cellular cAMP and [Ca(2+)]i in keratinocytes and fibroblasts. In contrast, PTHrP (107-139) increased [Ca(2+)]i but not cAMP in the two cell types. PTHrP (67-89) had no effect in keratinocytes, and only increased [Ca(2+)]i in fibroblasts. Vitamin D deficiency in weaned rats increased the expression of PTHrP mRNA in keratinocytes, and decreased it in fibroblasts and kidneys. Vitamin D deficiency increased PTH-1R mRNA expression in keratinocytes and kidneys, but not in fibroblasts. Although keratinocytes and skin fibroblasts are target cells for PTHrP and express PTH-1R, the two adjacent cell types differ as regards their intracellular signaling in response to PTHrP peptides. Moreover vitamin D regulates PTHrP and PTH-1R in a cell-specific manner.     

Parathyroid hormone secretion does not respond to changes of free calcium in electropermeabilized bovine parathyroid cells, but is stimulated with phorbol ester and cyclic AMP

The secretion of parathyroid hormone (PTH) is suppressed in bovine parathyroid cells by raised extracellular [Ca2+], and 12-0-tetradecanoyl-phorbol-13-acetate (TPA) stimulates the release of PTH from cells suppressed by high extracellular [Ca2+]. Extracellular and cytosolic free [Ca2+] are proportionally related in intact cells. To assess the role of cytosolic free [Ca2+] on PTH secretion, bovine parathyroid cells were rendered permeable by brief exposure to an intense electric field. PTH secretion was comparable at 40 nM, 500 nM, 5 microM, 28 microM, 0.5 mM and 2 mM [Ca2+] (release of total cellular PTH 3.7 +/- 0.5%, 3.9 +/- 0.4%, 3.4% +/- 0.3%, 3.9 +/- 0.4%, 3.1 +/- 0.3%, 3.5 +/- 0.7%, respectively), but the secretion was stimulated twofold (P less than 0.05 vs. control) in a dose and ATP dependent manner with TPA (100 nM) and cyclic AMP (1 mM). As a result, free [Ca2+] in the range of those observed in intact cells during regulation of PTH secretion by changes of extracellular [Ca2+] did not affect the release of PTH in permeabilized cells. The [Ca2+] independent stimulation of PTH release by TPA and cyclic AMP indicates that changes of cytosolic free [Ca2+] may represent a secondary event not related to the regulation of PTH secretion.     

Type V collagen-induced upregulation of capn2 (large subunit of m-calpain) gene expression and DNA fragmentation in 8701-BC breast cancer cells

Type V collagen is known to be over-deposited in the stroma of ductal infiltrating carcinomas of the breast. When used as a substrate, type V collagen restrains growth and invasion, and affects gene expression of 8701-BC ductal infiltrating carcinomas cells. Here we supplement existing data by demonstrating type V collagen dependent upregulation of capn2 gene expression in 8701-BC cells through differential display-PCR and Western blot assays. Furthermore, we suggest that our data obtained by centrifugal sedimentation and electrophoresis strongly suggest a correlation between calpain overproduction and DNA fragmentation, since the incubation with calpain inhibitor partly reverts the latter.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.     

Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism

Activation of the extracellular calcium-sensing receptor (CaR) stimulates mitogen-activated protein kinases to upregulate the synthesis and secretion of parathyroid hormone related peptide (PTHrP) from cells expressing the CaR heterologously or endogenously. The current experiments demonstrate that this occurs because CaR activation "transactivates" the EGF receptor (EGFR). Time dependent increases in tyrosine phosphorylation of the EGFR after addition of extracellular calcium ([Ca2+]o, 3 mM) occurred in stably CaR-transfected HEK293 cells but not in non-transfected HEK293 cells. AG1478, an EGFR kinase inhibitor, prevented the CaR-mediated increases of pERK and PTHrP release, while AG1296, a PDGFR kinase inhibitor, had no effect. Inhibitors of matrix metalloproteinase and heparin bound-EGF prevented the CaR-mediated increases of pERK and PTHrP, consistent with a "triple-membrane-spanning signaling" requirement for transactivation of the EGFR by the CaR. Proximal and distal signal transduction cascades activated by the CaR may reflect transactivation of the EGFR by the extracellular calcium-sensing receptor.     

Direct adhesion to type I and homotrimer collagens by breast carcinoma and embryonic epithelial cells in culture: a comparative study

A continuous cell line of neoplastic cells derived from ductal infiltrating carcinoma of the human breast (8701-BC), was assayed for its ability to adhere to collagen substrates. The collagens used were regular type I and type I homotrimer isolated from primary breast carcinomas. Comparative studies were performed using an embryonic epithelial cell line derived from human intestine (Int. 407). The neoplastic cells adhere equally well to both collagens, while the embryonic epithelial cells recognized only the homotrimer. Some receptor diversity was recognized in the adhesion of the two cell lines to homotrimer collagen. The data demonstrate a functional difference between type I and homotrimer collagen with regard to cellular recognition and attachment. In addition, the data suggest that oncogenic transformation of breast epithelial cells promotes their adhesive properties to interstitial collagens and that this may be relevant to their increased potential to invade host tissue.     

Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells

The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.