MUC1 and Polarity Markers INADL and SCRIB Identify Salivary Ductal Cells.

Current treatments for xerostomia/dry mouth are palliative and largely ineffective. A permanent clinical resolution is being developed to correct hyposalivation using implanted hydrogel-encapsulated salivary human stem/progenitor cells (hS/PCs) to restore functional salivary components and increase salivary flow. Pluripotent epithelial cell populations derived from hS/PCs, representing a basal stem cell population in tissue, can differentiate along either secretory acinar or fluid-transporting ductal lineages. To develop tissue-engineered salivary gland replacement tissues, it is critical to reliably identify cells in tissue and as they enter these alternative lineages. The secreted protein α-amylase, the transcription factor MIST1, and aquaporin-5 are typical markers for acinar cells, and K19 is the classical ductal marker in salivary tissue. We found that early ductal progenitors derived from hS/PCs do not express K19, and thus earlier markers were needed to distinguish these cells from acinar progenitors. Salivary ductal cells express distinct polarity complex proteins that we hypothesized could serve as lineage biomarkers to distinguish ductal cells from acinar cells in differentiating hS/PC populations. Based on our studies of primary salivary tissue, both parotid and submandibular glands, and differentiating hS/PCs, we conclude that the apical marker MUC1 along with the polarity markers INADL/PATJ and SCRIB reliably can identify ductal cells in salivary glands and in ductal progenitor populations of hS/PCs being used for salivary tissue engineering. Other markers of epithelial maturation, including E-cadherin, ZO-1, and partition complex component PAR3, are present in both ductal and acinar cells, where they can serve as general markers of differentiation but not lineage markers.

Medical and Dental Electronic Health Record Reporting Discrepancies in Integrated Patient Care.

INTRODUCTION: Oral health mirrors systemic health; yet, few clinics worldwide provide dental care as part of primary medical care, nor are dental records commonly integrated with medical records. OBJECTIVES: To determine the degree to which misreporting of underlying health conditions poses problems for dental clinicians, we assessed misreporting of 2 common medical health conditions-hypertension and diabetes-at the time of dental examination and assessment. METHODS: Using comparative chart analysis, we analyzed medical records of a diverse group of patients previously seen at the University of Texas Physician outpatient practice and then treated at the University of Texas Health Science Center at Houston School of Dentistry. Electronic health records of patients aged ≥18 y were extracted from 2 databases: Allscripts (University of Texas Physician) and axiUm (University of Texas Health Science Center at Houston). We identified 1,013 patients with the commonly occurring conditions of diabetes, hypertension, or both, with nonintegrated records contained in Allscripts and axiUm. We identified the percentage of those patients previously diagnosed with diabetes and/or hypertension by their physicians who failed to report these conditions to their dental clinicians. RESULTS: Of those patients with diabetes, 15.1% misreported their diabetes condition to their dental clinicians, while 29.0% of patients with hypertension also misreported. There was no relationship between sex and misreporting of hypertension or diabetes, but age significantly affected reporting of hypertension, with misreporting decreasing with age. CONCLUSIONS: Because these conditions affect treatment planning in the dental clinic, misreporting of underlying medical conditions can have negative outcomes for dental patients. We conclude that policies that support the integration of medical and dental records would meaningfully increase the quality of health care delivered to patients, particularly those dental patients with underlying medical conditions. KNOWLEDGE TRANSFER STATEMENT: Our study illustrates an urgent need for policy innovation within a currently fragmented health care delivery system. Dental clinicians rely on the accuracy of health information provided by patients, which we found was misreported in ~15% to 30% of dental patient records. An integrated health care system can close these misreporting gaps. Policies that support the integration of medical and dental records can improve the quality of health care delivered, particularly for dental patients with underlying medical conditions.

Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells.

Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the development of this promising gene therapy continues, safety considerations are a high priority, particularly those that remove nonhuman agents (i.e., viral vectors and genetic sequences of bacterial origin). In this study, we applied 2 emerging technologies, artificial transcriptional complexes and epigenetic editing, to explore whether AQP1 expression could be achieved by activating the native gene locus in a human salivary ductal cell line and primary salivary human stem/progenitor cells (hS/PCs), as opposed to the conventional approach of cytomegalovirus promoter-driven expression from an episomal vector. In our first study, we used a cotransfection strategy to express the components of the dCas9-SAM system to create an artificial transcriptional complex at the AQP1 locus in A253 and hS/PCs. We found that AQP1 expression was induced at a magnitude comparable to adenoviral infection, suggesting that AQP1 is primarily silenced through pretranscriptional mechanisms. Because earlier literature suggested that pretranscriptional silencing of AQP1 in salivary glands is mediated by methylation of the promoter, in our second study, we performed global, chemical demethylation of A253 cells and found that demethylation alone induced robust AQP1 expression. These results suggest the potential for success by inducing AQP1 expression in human salivary ductal cells through epigenetic editing of the native promoter.

IL-1ß induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

Chronic inflammation is associated with advanced prostate cancer (PCa), although the mechanisms governing inflammation-mediated PCa progression are not fully understood. PCa progresses to an androgen independent phenotype that is incurable. We previously showed that androgen independent, androgen receptor negative (AR(-) ) PCa cell lines have high p62/SQSTM1 levels required for cell survival. We also showed that factors in the HS-5 bone marrow stromal cell (BMSC) conditioned medium can upregulate p62 in AR(+) PCa cell lines, leading us to investigate AR expression under those growth conditions. In this paper, mRNA, protein, and subcellular analyses reveal that HS-5 BMSC conditioned medium represses AR mRNA, protein, and nuclear accumulation in the C4-2 PCa cell line. Using published gene expression data, we identify the inflammatory cytokine, IL-1ß, as a candidate BMSC paracrine factor to regulate AR expression and find that IL-1ß is sufficient to both repress AR and upregulate p62 in multiple PCa cell lines. Immunostaining demonstrates that, while the C4-2 population shows a primarily homogeneous response to factors in HS-5 BMSC conditioned medium, IL-1ß elicits a strikingly heterogeneous response; suggesting that there are other regulatory factors in the conditioned medium. Finally, while we observe concomitant AR loss and p62 upregulation in IL-1ß-treated C4-2 cells, silencing of AR or p62 suggests that IL-1ß regulates their protein accumulation through independent pathways. Taken together, these in vitro results suggest that IL-1ß can drive PCa progression in an inflammatory microenvironment through AR repression and p62 induction to promote the development and survival of androgen independent PCa.

Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior.

Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.

TISSUE ENGINEERING PERFUSABLE CANCER MODELS.

The effect of fluid flow on cancer progression is currently not well understood, highlighting the need for perfused tumor models to close this gap in knowledge. Enabling biological processes at the cellular level to be modeled with high spatiotemporal control, microfluidic tumor models have demonstrated applicability as platforms to study cell-cell interactions, effect of interstitial flow on tumor migration and the role of vascular barrier function. To account for the multi-scale nature of cancer growth and invasion, macroscale models are also necessary. The consideration of fluid dynamics within tumor models at both the micro- and macroscopic levels may greatly improve our ability to more fully mimic the tumor microenvironment.

Mecp2 deficiency decreases bone formation and reduces bone volume in a rodent model of Rett syndrome.

Rett syndrome (RTT), a neurological disorder characterized by neurological impairment and a high frequency of osteopenia which often manifests early in childhood, most often is caused by inactivating mutations in the X-linked gene encoding a regulator of epigenetic gene expression, methyl CpG binding protein, MeCP2. Clinical data show that, along with neurological defects, females with RTT frequently have marked decreases in bone mineral density (BMD) beyond that expected from disuse atrophy. To investigate the relationship between loss of Mecp2 and reduced BMD, we used a Mecp2 null mouse model, Mecp2 (-/yBIRD), for our histological and biochemical studies. Mecp2 (-/yBIRD) mice have significantly shorter femurs and an overall reduced skeletal size compared to wild-type mice by post-natal day 60 (P60). Histological and histomorphometric studies identified growth plate abnormalities as well as decreased cortical and trabecular bone in P21 and especially in P60 Mecp2 (-/yBIRD) mice. Dynamic histomorphometry revealed decreased mineral apposition rates (MAR) in Mecp2 null femoral trabecular bone as well as in calvarial bone samples. While changes in MAR of cortical bone were not significant, loss of Mecp2 significantly reduced cortical, trabecular and calvarial bone volume compared with age-matched wild-type animals. These differences indicate that Mecp2 deficiency leads to osteoblast dysfunction, which translates into reduced osteoid deposition accounting for the reduced bone volume phenotype. While individual variations were observed in OPG and Rankl concentrations, molar ratios of OPG:Rankl at P21 and P60 were comparable between wild-type and Mecp2 (-/yBIRD) mice and showed a consistent excess of OPG. In tibial sections, TRAP staining demonstrated equivalent osteoclast number per bone surface measurements between wild-type and null animals. Our work with a Mecp2 null mouse model suggests epigenetic regulation of bone in the Mecp2 (-/yBIRD) mice which is associated with decreased osteoblast activity rather than increased osteoclastic bone loss.

Heparanase expression and activity influences chondrogenic and osteogenic processes during endochondral bone formation.

Endochondral bone formation is a highly orchestrated process involving coordination among cell-cell, cell-matrix and growth factor signaling that eventually results in the production of mineralized bone from a cartilage template. Chondrogenic and osteogenic differentiation occur in sequence during this process, and the temporospatial patterning clearly requires the activities of heparin binding growth factors and their receptors. Heparanase (HPSE) plays a role in osteogenesis, but the mechanism by which it does so is incompletely understood. We used a combination of ex vivo and in vitro approaches and a well described HPSE inhibitor, PI-88 to study HPSE in endochondral bone formation. In situ hybridization and immunolocalization with HPSE antibodies revealed that HPSE is expressed in the peri-chondrium, peri-osteum, and at the chondro-osseous junction, all sites of key signaling events and tissue morphogenesis. Transcripts encoding Hpse also were observed in the pre-hypertrophic zone. Addition of PI-88 to metatarsals in organ culture reduced growth and suggested that HPSE activity aids the transition from chondrogenic to osteogenic processes in growth of long bones. To study this, we used high density cultures of ATDC5 pre-chondrogenic cells grown under conditions favoring chondrogenesis or osteogenesis. Under chondrogenic conditions, HPSE/Hpse was expressed at high levels during the mid-culture period, at the onset of terminal chondrogenesis. PI-88 addition reduced chondrogenesis and accelerated osteogenesis, including a dramatic up-regulation of osteocalcin levels. In normal growth medium, addition of PI-88 reduced migration of ATDC-5 cells, suggesting that HPSE facilitates cartilage replacement by bone at the chondro-osseous junction by removing the HS component of proteoglycans, such as perlecan/HSPG2, that otherwise prevent osteogenic cells from remodeling hypertrophic cartilage.

Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds.

Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17 , 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.

Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells.

We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)(2)D(3) and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D(3)-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)(2)D(3) binding, but did not affect binding to the nuclear receptor for 1,25(OH)(2)D(3). Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D(3)-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)(2)D(3) in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D(3)-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D(3)-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.

Rodent osteoblastic cells express voltage-sensitive calcium channels lacking a gamma subunit.

Voltage-sensitive calcium channels (VSCC) open in response to external stimuli, including calcitropic hormones, that alter plasma membrane calcium (Ca2+) permeability. Ca2+ that enters the cell through these channels serves a second messenger function, eliciting cellular responses that include secretion and changes in gene expression. In osteoblasts, VSCCs serve as key regulators of Ca2+ permeability and are a major class of calcitropic hormone-sensitive Ca2+ channels present in the plasma membrane. The members of the VSCC family exist as a complex of polypeptide subunits that are comprised of a pore-forming alpha1 subunit, an intracellular beta subunit, a dimer of disulfide-linked alpha2 and delta subunits, and in some tissues, a gamma subunit. Previous studies in our laboratory have shown that the major functional alpha1 subunit present in osteoblasts is the alpha1C (CaV1.2). To determine the complement of auxiliary subunits present in rodent osteoblastic cells, we employed RT-PCR using a battery of subunit specific primers and appropriate tissue controls. Immunohistochemistry also was performed, using available subunit specific antibodies, to measure protein expression and localization. Cell types examined included MC3T3-E1 at various stages of differentiation, ROS 17/2.8 osteosarcoma, and primary cultures of rat calvarial osteoblasts. The results indicate that all cells expressed multiple beta subunit classes and alpha2delta dimers, but no gamma subunits, regardless of differentiation state. We propose a structure for the functional osteoblast VSCC that consists of alpha1, beta, alpha2delta subunits and is devoid of a gamma subunit.

Membrane receptors for vitamin D steroid hormones: potential new drug targets.

There is increasing evidence that steroid hormones derived from vitamin D act through classical nuclear receptors (nVDR), as well as specific binding sites on the plasma membrane of target cells that are coupled to signal transduction systems. These sites are referred to as Membrane Associated, Rapid Response Steroid (MARRS) binding proteins or complexes. In the case of the seco-steroid 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the two 'receptors' appear to be different proteins with distinct affinities for vitamin D analogs. These differences may be useful in a number of clinical situations. In treating leukemias, it would be useful to promote the actions of the nVDR for differentiation to macrophages while blocking the 1,25D3-MARRS in intestine, which might contribute to the undesirable side effect of hypercalcemia. In contrast, stimulation of the intestinal 1,25D3-MARRS would be desirable in the elderly, since this signalling system appears to decline with age in model systems, potentially contributing to diminished intestinal absorption of calcium and associated bone loss. Bone itself is known to have osteoblasts that respond to 1,25(OH)2D3 through both nVDR and 1,25D3-MARRS mechanisms. Both systems are required for bone-building activities. Osteoclasts lack the nVDR, but may become activated through the 1,25D3-MARRS, offering another site of drug intervention in the treatment of osteoporosis. Finally, during tooth mineralization, immunohistochemical studies reveal an absence of the nVDR and a marked appearance of the 1,25D3-MARRS. In addition to our growing knowledge of 1,25(OH)2D3, the physiological actions of a lesser studied metabolite of vitamin D, 24,25(OH)2D3, are coming to light and may offer additional targets for pharmaceutical modulation.

Heparan sulfate interacting protein (HIP/L29) negatively regulates growth responses to basic fibroblast growth factor in gingival fibroblasts.

Basic fibroblast growth factor (bFGF) modulates gingival growth, and its release from heparan sulfate (HS) in the extracellular matrix (ECM) governs local tissue bioavailability. We identified a heparin/HS interacting protein (HIP/L29) that recognizes specific HS sequences. We hypothesize that HIP/L29, by modulating the interactions of bFGF with HS chains on proteoglycans, could regulate bFGF bioavailability. To investigate interactions between bFGF and HIP/L29, we isolated and cultured fibroblasts from normal gingiva and overgrown gingiva from patients on cyclosporine (CSA). bFGF significantly stimulated gingival fibroblast proliferation with or without heparin. Recombinant human HIP/L29 dramatically decreased bFGF-induced proliferation, but did not alter responses to insulin-like growth factor-1 (IGF-1). Analysis of mitogen-activated protein kinase (MAPK) phosphorylation patterns showed that bFGF stimulation of p44 (Erk-1), but not p42 (Erk-2), also was inhibited by HIP/L29 in a dose-dependent manner. Together, these results support our hypothesis that HIP/L29 modulates the bioavailability and action of bFGF.

Perlecan: an important component of the cartilage pericellular matrix.

Perlecan (Pln) is a large proteoglycan that can bear HS (heparan sulfate) and chondroitin sulfate glycosaminoglycans. Previous studies have demonstrated that Pln can interact with growth factors and cell surfaces either via its constituent glycosaminoglycan chains or core protein. Herein, we summarize studies demonstrating spatially and temporally regulated expression of Pln mRNA and protein in developing and mature cartilage. Mutations either in the Pln gene or in genes involved in glycosaminoglycan assembly result in severe cartilage phenotypes seen in both human syndromes and mouse model systems. In vitro studies demonstrate that Pln can trigger chondrogenic differentiation of multipotential mouse CH310T1/2 stem cells as well as maintain the phenotype of adult human chondrocytes. Structural mapping indicates that these activities lie entirely within domain I, a region unique to Pln, and that they require glycosaminoglycans. We also discuss data indicating that Pln cooperates with the key chondrogenic growth factor, BMP-2, to promote expression of hypertrophic chondrocyte markers. Collectively, these studies indicate that Pln is an important component of human cartilage and may have useful applications in tissue engineering and cartilage-directed therapeutics.

Type I collagen-mediated proliferation of PC3 prostate carcinoma cell line: implications for enhanced growth in the bone microenvironment.

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to bone. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as, extracelluar matrix components, have been implicated in the spread and propagation of prostatic carcinoma. The prostate cell line, PC3, adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the alpha(1), alpha(2) and alpha(3) collagen binding integrin subunits. Antibody function blocking studies reveal that PC3 cells can utilize alpha(2)beta(1) and alpha(3)beta(1) integrins to adhere to collagen I. Cells plated on collagen I exhibit increased rates of proliferation over cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of cyclin D1, a molecule associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), map kinase (MAPK) and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. Type I collagen may facilitate the colonization and growth of metastatic prostate tumor cells in the bone microenvironment.

Parathyroid hormone-stimulated resorption in calvaria cultured in serum-free medium is enhanced by the calcium-mobilizing activity of 1,25-dihydroxyvitamin D(3).

1,25(OH)(2)D(3) enhances parathyroid hormone (PTH)-induced Ca(2+) signaling in osteoblasts by activating plasma membrane voltage-sensitive Ca(2+) channels (VSCCs). The ability of 1,25(OH)(2)D(3) or the VSCC-activating analog AT (25-hydroxy-16-ene-23-yne-D(3)) to enhance parathyroid hormone-stimulated (45)Ca(2+) release from cultured new-born rat calvaria was measured. Analog BT (1,24-dihydroxy-22-ene-24-cyclopropyl-D(3)), that does not mobilize Ca(2+), also was tested along with PTH. Control experiments were performed with and without PTH and with and without serum. Individual calvaria labeled in utero with (45)Ca(2+) were cultivated in serum-free medium on filters at the medium/air interface of 24-well culture plates and (45)Ca(2+) release followed over 72 h. The results demonstrated that 1,25(OH)(2)D(3) and the Ca(2+)-mobilizing analog, AT, but not the nuclear receptor-binding analog, BT, enhanced PTH-stimulated (45)Ca(2+) release under serum-free conditions. This enhancement effect of the seco-steroids was not evident in the presence of 10% fetal calf serum. The effect of analog AT was faster than that of 1,25(OH)(2)D(3). Nitrendipine, a specific L-type VSCC blocker, attenuated enhancement by vitamin D compounds, indicating that the high-threshold L-type VSCC is a molecular transducer of costimulation. These results emphasize the synergy between the calcitropic hormones 1,25(OH)(2)D(3) and PTH in cultures containing osteoblasts and osteoclasts, and suggest that the Ca(2+)-mobilizing activity of 1,25(OH)(2)D(3) enhances Ca(2+) release from bone.

Integrin expression and usage by prostate cancer cell lines on laminin substrata.

During prostate cancer progression, invasive glandular epithelial cells move out of the ductal-acinar architecture and through the surrounding basement membrane. Extracellular matrix proteins and associated soluble factors in the basal lamina and underlying stroma are known to be important regulators of prostate cell behaviors in both normal and malignant tissues. In this study, we assessed cell interactions with extracellular matrix and stromal factors during disease progression by characterizing integrin usage and expression in a series of parental and lineage-derived LNCaP human prostate cancer cell lines. Although few shifts in integrin expression were found to accompany disease progression, integrin heterodimer usage did change significantly. The more metastatic sublines were distinct in their use of alphavbeta3 and, when compared with parental LNCaP cells, showed a shift in alpha6 heterodimerization, a subunit critical not only for interaction with prostate basal lamina but also for interaction with the bone matrix, a favored site of prostate cancer metastases.

Bioactive analogs that simulate subsets of biological activities of 1alpha,25(OH)(2)D(3) in osteoblasts.

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] treatment of osteoblastic cells elicits a series of measurable responses that include both rapid, membrane-initiated effects and longer-term nuclear receptor-mediated effects. Structural analogs have been identified and characterized that selectively activate subsets of these pathways. Two analogs from over 35 that have been tested were chosen for this comparison because they activate non-overlapping response pathways, presumably representing either membrane-initiated or nuclear receptor-initiated activities. Compound AT [25(OH)-16ene-23yne-D(3)] lacks the 1-hydroxyl essential for interacting with the nuclear receptor, but triggers Ca(2+) influx through plasma membrane Ca(2+) channels, augments parathyroid hormone (PTH)-induced Ca(2+) signals, dephosphorylates the matrix protein osteopontin (OPN), and along with PTH stimulates release of calcium from calvaria in organ culture. Compound BT [1alpha,24(OH)(2)-22ene-24cyclopropyl-D(3)] does not elicit any of the rapid responses or enhance PTH-induced bone resorption, but binds to the nuclear receptor for 1alpha,25(OH)(2)D(3) and increases steady state mRNA levels of both OPN and osteocalcin over a 48 h period. Together, these two analogs recapitulate all of the known actions of 1alpha,25(OH)(2)D(3) on osteoblasts. Based on these findings, we conclude that Ca(2+) release from bone stimulated by 1alpha,25(OH)(2)D(3) and PTH is related to the rapid, membrane-initiated actions and is not likely to involve binding to the nuclear receptor for 1alpha,25(OH)(2)D(3). Longer term stimulation of bone formation by 1alpha,25(OH)(2)D(3), however, appears to involve solely the nuclear receptor-mediated effects. These findings support our model of 1alpha,25(OH)(2)D(3) as a coupling factor for bone resorption and formation during bone remodeling.

1,25 dihydroxyvitamin D(3) activates sphingomyelin turnover in ROS17/2.8 osteosarcoma cells without sphingolipid-induced changes in cytosolic Ca(2+).

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] initiates the hydrolysis of sphingomyelin in ROS 17/2.8 osteosarcoma cells with the resultant generation of cell-associated ceramide. Increases in ceramide levels were detectable at 15 min and maximal one hour after exposure of cells to 1,25(OH)(2)D(3). Neither 1,25(OH)(2)D(3) nor exogenous ceramide elicited a change in cytosolic free Ca(2+) ([Ca(2+)](i)). Transient elevations in [Ca(2+)](i) were observed when cells were exposed to exogenous sphingosine, but there was no detectable conversion of ceramide to sphingosine in 1, 25(OH)(2)D(3)-treated cells. Ceramide also did not stimulate Ca(2+) uptake across ROS 17/2.8 cell plasma membranes. Collectively, these results suggest that 1,25(OH)(2)D(3) activates sphingomyelin turnover in ROS 17/2.8 osteosarcoma cells but that the sphingolipid metabolite ceramide is not responsible for 1,25(OH)(2)D(3)-induced activation of plasma membrane Ca(2+) channels.

Ribozyme ablation demonstrates that the cardiac subtype of the voltage-sensitive calcium channel is the molecular transducer of 1, 25-dihydroxyvitamin D(3)-stimulated calcium influx in osteoblastic cells.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) stimulates transmembrane influx of Ca(2+) through L-type voltage-sensitive Ca(2+) channels (VSCCs) in ROS 17/2.8 osteoblastic cells. Ca(2+) influx modulates osteoblastic activities including matrix deposition, hormone responsiveness, and Ca(2+)-dependent signaling. 1, 25(OH)(2)D(3) also regulates transcript levels encoding VSCCs. L-type VSCCs are multisubunit complexes composed of a central pore-forming alpha(1) subunit and four additional subunits. The alpha(1) subunit is encoded by one gene in a multimember family, defining tissue-specific subtypes. Osteoblasts synthesize two splice variants of the alpha(1C) cardiac VSCC subtype; however, the molecular identity of the 1,25(OH)(2)D(3)-regulated VSCC remained unknown. We created a ribozyme specifically cleaving alpha(1C) mRNA. To increase target ablation efficiency, the ribozyme was inserted into U1 small nuclear RNA (snRNA) by engineering the U1 snRNA expression cassette, conferring the ribozyme transcript with stabilizing stem-loops at both sides and the Sm binding site that facilitates localization into nucleoplasm. After transfection of ROS 17/2.8 cells with U1 ribozyme-encoding vector, stable clonal cells were selected in which the expression of alpha(1C) transcript and protein were strikingly reduced. Ca(2+) influx assays in ribozyme transfectants showed selective attenuation of depolarization and 1, 25(OH)(2)D(3)-regulated Ca(2+) responses. We conclude that the cardiac subtype of the L-type VSCC is the transducer of stimulated Ca(2+) influx in ROS 17/2.8 osteoblastic cells.