Vitamin D3 Regulates the Formation and Degradation of Gap Junctions in Androgen-Responsive Human Prostate Cancer Cells

1α-25(OH)₂ vitamin D₃ (1-25D), an active hormonal form of Vitamin D₃, is a well-known chemopreventive and pro-differentiating agent. It has been shown to inhibit the growth of several prostate cancer cell lines. Gap junctions, formed of proteins called connexins (Cx), are ensembles of cell-cell channels, which permit the exchange of small growth regulatory molecules between adjoining cells. Cell-cell communication mediated by gap junctional channels is an important homeostatic control mechanism for regulating cell growth and differentiation. We have investigated the effect of 1-25D on the formation and degradation of gap junctions in an androgen-responsive prostate cancer cell line, LNCaP, which expresses retrovirally-introduced Cx32. Connexin32 is expressed by the luminal and well-differentiated cells of normal prostate and prostate tumors. Our results document that 1-25D enhances the expression of Cx32 and its subsequent assembly into gap junctions. Our results further show that 1-25D prevents androgen-regulated degradation of Cx32, post-translationally, independent of androgen receptor (AR)-mediated signaling. Finally, our findings document that formation of gap junctions sensitizes Cx32-expressing LNCaP cells to the growth inhibitory effects of 1-25D and alters their morphology. These findings suggest that the growth-inhibitory effects of 1-25D in LNCaP cells may be related to its ability to modulate the assembly of Cx32 into gap junctions.     

Androgen-regulated formation and degradation of gap junctions in androgen-responsive human prostate cancer cells

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

Photoisomerization of retinoic acid and its influence on regulation of human keratinocyte growth and differentiation

Retinoic acid constantly undergoes structural inter-conversions among the geometrical isomers (all-trans-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid and 9-13-di-cis-retinoic acid) by photoisomerization under natural light. Geometric isomers of retinoic acid thus formed showed different effects on human epidermal keratinocyte growth and differentiation. The ability of the isomers to inhibit the synthesis of cornified envelope (terminal event in the keratinocyte differentiation program) changed rapidly when illuminated by white fluorescent light. The 11-cis-retinoic acid had a 3-fold stronger activity to inhibit the growth of keratinocytes than the other geometric isomers. On the other hand, all-trans-retinoic acid, 9-cis-retinoic acid and 9-13-di-cis-retinoic acid exhibited a 3-fold greater ability to inhibit synthesis of involucrin, transglutaminase and the cornified envelopes. The regulation of keratin expression by the geometric isomers of retinoic acids was extremely complex. Level of keratin-1 (K1) mRNA was increased by 11-cis-retinoic acid and 13-cis-retinoic acid, but suppressed by 9,13-di-cis-retinoic acids while all-trans-retinoic acid and 9-cis-retinoic acid had no effect. Level of keratin-10 (K10) mRNA was strongly inhibited by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid as compared to 13-cis-retinoic acid and 9,13-di-cis-retinoic acids. The mRNA level of keratin-14 (K14) was suppressed by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid but not influenced by 13-cis-retinoic acid and 9,13-di-cis-retinoic acid. Natural light induced structural inter-conversions among the geometric isomers of retinoic acids in tissues-especially the skin, might play a crucial role in the regulation of growth and differentiation of keratinocytes.     

Specificity in the synergism between retinoic acid and EGF on the growth of adult human skin fibroblasts

Vitamin A (retinol) and five retinoids were tested for their ability to enhance epidermal growth factor (EGF) stimulation of adult human skin fibroblast growth in vitro. The retinoids utilized in this study were RO-1-5488 (all-trans-retinoic acid), RO-4-3780 (13-cis-retinoic acid), RO-10-9359, RO-10-1670, and RO-21-6583. Retinol and each retinoid were capable of stimulating fibroblast growth alone (0-86%), while 13-cis and all-trans-retinoic acid were the most potent in potentiating the EGF promotion of fibroblast growth. Other growth factors tested in addition to EGF were nerve growth factor (NGF), fibroblast growth factor (FGF), and thrombin. While EGF and FGF stimulated fibroblast growth to the same degree (2.3-fold), only growth stimulated by EGF was potentiated by retinoic acid. Since retinoic acid might enhance the EGF stimulation of cell growth by increasing either EGF receptor number or binding affinity, the binding of 125I-labeled EGF was carried out in the presence of retinoic acid and the data were subjected to a Scatchard-type analysis. No change in EGF receptor number or affinity was seen in the presence of retinoic acid. The data indicate a specific interaction between retinoid acid and EGF which results in the potentiation of the EGF-stimulated cell growth. Furthermore, the mechanism of this interaction does not seem to involve the initial binding of EGF to its plasma membrane receptor or the available number of EGF receptors located on the cell surface.     

Families of retinoid dehydrogenases regulating vitamin A function: production of visual pigment and retinoic acid.

Vitamin A (retinol) and provitamin A (beta-carotene) are metabolized to specific retinoid derivatives which function in either vision or growth and development. The metabolite 11-cis-retinal functions in light absorption for vision in chordate and nonchordate animals, whereas all-trans-retinoic acid and 9-cis-retinoic acid function as ligands for nuclear retinoic acid receptors that regulate gene expression only in chordate animals. Investigation of retinoid metabolic pathways has resulted in the identification of numerous retinoid dehydrogenases that potentially contribute to metabolism of various retinoid isomers to produce active forms. These enzymes fall into three major families. Dehydrogenases catalyzing the reversible oxidation/reduction of retinol and retinal are members of either the alcohol dehydrogenase (ADH) or short-chain dehydrogenase/reductase (SDR) enzyme families, whereas dehydrogenases catalyzing the oxidation of retinal to retinoic acid are members of the aldehyde dehydrogenase (ALDH) family. Compilation of the known retinoid dehydrogenases indicates the existence of 17 nonorthologous forms: five ADHs, eight SDRs, and four ALDHs, eight of which are conserved in both mouse and human. Genetic studies indicate in vivo roles for two ADHs (ADH1 and ADH4), one SDR (RDH5), and two ALDHs (ALDH1 and RALDH2) all of which are conserved between humans and rodents. For several SDRs (RoDH1, RoDH4, CRAD1, and CRAD2) androgens rather than retinoids are the predominant substrates suggesting a function in androgen metabolism as well as retinoid metabolism.     

Regulation of gap junctions by phosphorylation of connexins

Gap junctions are a unique type of intercellular junction found in most animal cell types. Gap junctions permit the intercellular passage of small molecules and have been implicated in diverse biological processes, such as development, cellular metabolism, and cellular growth control. In vertebrates, gap junctions are composed of proteins from the "connexin" gene family. The majority of connexins are modified posttranslationally by phosphorylation, primarily on serine amino acids; however, phosphotyrosine has also been detected in connexin from cells coexpressing nonreceptor tyrosine protein kinases. Connexins are targeted by numerous protein kinases, of which some have been identified: protein kinase C, mitogen-activated protein kinase, and the v-Src tyrosine protein kinase. Phosphorylation has been implicated in the regulation of a broad variety of connexin processes, such as the trafficking, assembly/disassembly, degradation, as well as the gating of gap junction channels. This review examines the consequences of connexin phosphorylation for the regulation of gap junctional communication.     

Clinical applications of retinoids in cancer medicine

The retinoids are compounds structurally related to vitamin A. The most extensively studied agents in cancer medicine include all-trans-retinoic acid, 9-cis-retinoic acid, and 13-cis-retinoic acid. In addition to several described immune regulatory functions, these agents may exert their antineoplastic effects through the regulation of tumor suppressor genes such as RAR-beta2. The survival benefit provided to patients with acute promyelocytic leukemia (APL) after induction therapy with all-trans RA and the responses experienced by patients with cutaneous lesions from Kaposi's sarcoma and cutaneous T cell lymphoma treated with 9-cis RA and a selective rexinoid--bexarotene--respectively, led to their approval by the Food and Drug Administration during the last decade. As chemopreventive agents, retinoids have proven to effectively regress laryngeal papillomatosis and oral leukoplakia lesions. The ability of 13-cis-RA to prevent second primary malignancies in patients with carcinoma of the head and neck has also been demonstrated. Unfortunately, this intervention did not affect the primary tumor recurrence rates. The toxicity and efficacy of retinoids administered in combination with other biological and cytotoxic agents have also been explored in patients with renal cell carcinoma, breast cancer, myelodysplasia, prostate, cervix, and other malignancies with a broad range of reported responses. Further characterization of the molecular processes modulated by these agents will serve to better define their role in the prevention and treatment of human cancer and to tailor specific targeted therapies in combination with other compounds. Newer and more selective retinoids and rexinoids are completing phase I and phase II studies and hold promising.     

Improved synthesis of histone deacetylase inhibitors (HDIs) (MS-275 and CI-994) and inhibitory effects of HDIs alone or in combination with RAMBAs or retinoids on growth of human LNCaP prostate cancer cells and tumor xenografts

We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors (HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively. The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease in the S phase in LNCaP cells. 2+SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differentiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2+SAHA were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.     

Microinjection of cultured rat embryos: studies with retinol, 13-cis- and all-trans-retinoic acid

Retinol, all-trans-retinoic acid or 13-cis-retinoic acid were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships of the retinoids showed that the dysmorphogenic effects were qualitatively similar for all three, but were elicited by a low concentration of all-trans-retinoic acid (250 ng/ml), a 6- to 7-fold higher concentration of 13-cis-retinoic acid and an approximately 16-fold higher concentration of retinol. After microinjection of 2,000 ng/ml of retinol, no dysmorphogenesis was observed but instead an increase in all growth parameters as compared to the controls.     

Impaired trafficking of connexins in androgen-independent human prostate cancer cell lines and its mitigation by alpha-catenin

Gap junctions, composed of connexins, provide a pathway of direct intercellular communication for the diffusion of small molecules between cells. Evidence suggests that connexins act as tumor suppressors. We showed previously that expression of connexin-43 and connexin-32 in an indolent prostate cancer cell line, LNCaP, resulted in gap junction formation and growth inhibition. To elucidate the role of connexins in the progression of prostate cancer from a hormone-dependent to -independent state, we introduced connexin-43 and connexin-32 into an invasive, androgen-independent cell line, PC-3. Expression of these proteins in PC-3 cells resulted in intracellular accumulation. Western blot analysis revealed a lack of Triton-insoluble, plaque-assembled connexins. In contrast to LNCaP cells, connexins could not be cell surface-biotinylated and did not reside in the cell surface derived endocytic vesicles, in PC-3 cells, suggesting impaired trafficking to the cell surface. Intracellular accumulation of connexins was observed in several androgen-independent prostate cancer cell lines. Transient expression of alpha-catenin facilitated the trafficking of both connexins to the cell surface and induced gap junction assembly. Our results suggest that impaired trafficking, and not the inability to form gap junctions, is the major cause of communication deficiency in human prostate cancer cell lines.     

Retinaldehyde, a potent inhibitor of gap junctional intercellular communication

Retinaldehyde and retinoic acid are derivatives of vitamin A, and retinaldehyde is the precursor for the synthesis of retinoic acid, a well-known inhibitor of gap junctional intercellular communication. In this investigation, we asked the question if retinaldehyde has similar effects on gap junctions. Gap junctional intercellular communication was measured by scrape-loading and preloading dye-transfer methods, and studies were carried out mainly on cultured liver epithelial cells. Retinaldehyde was found to be a more potent inhibitor (dye transfer reduced by 50% at 2.8 μM) than retinoic acid (dye transfer reduced by 50% at 30 μM) and glycyrrhetinic acid (dye transfer reduced by 50% at 65 μM). Both the 11-cis and all-trans forms of retinaldehyde were equally effective. Retinaldehyde inhibited dye transfer of both anionic Lucifer yellow and cationic Neurobiotin. Inhibition by retinaldehyde developed in less than two minutes at 50 μM, but unlike the reported case with retinoic acid, recovery was slower, though full. In addition to liver epithelial cells, retinaldehyde inhibited gap junctional communication in lens epithelial cells, retinal pigment epithelial cells and retinal ganglion cells.     

The actions of retinoids on cellular growth correlate with their actions on gap junctional communication

Retinoic acid (a possible morphogen), its biological precursor retinol, and certain synthetic derivatives of retinol profoundly change junctional intercellular communication and growth (saturation density) in 10T 1/2 and 3T3 cells and in their transformed counterparts. The changes correlate: growth decreases as the steady-state junctional permeability rises, and growth increases as that permeability falls. Retinoic acid and retinol exert quite different steady-state actions on communication at noncytotoxic concentrations in the normal cells: retinoic acid inhibits communication at 10(-10)-10(-9) M and enhances at 10(-9)-10(-7) M, whereas retinol only enhances (10(-8)-10(-6) M). In v-mos-transformed cells the enhancement is altogether lacking. But regardless of the retinoid or cell type, all growth responses show essentially the same dependence on junctional permeability. This is the expected behavior if the cell-to-cell channels of gap junctions disseminate growth-regulating signals through cell populations.     

13-cis-retinoic acid metabolism in vivo. The major tissue metabolites in the rat have the all-trans configuration

The liver and intestinal metabolites of orally dosed 13-cis-[11-3H]retinoic acid were analyzed in normal and 13-cis-retinoic acid treated rats 3 h after administration of the radiolabeled retinoid. all-trans-Retinoic acid was identified as a liver and intestinal mucosa metabolite in normal rats given physiological doses of 13-cis-[3H]retinoic acid. all-trans-Retinoyl glucuronide was identified as the most abundant radiolabeled metabolite in mucosa and a prominent liver metabolite under the same conditions. Thus, the major 13-cis-retinoic acid metabolites retained in liver and mucosa, two retinoid target tissues, had the all-trans configuration. These data indicate that the isomerization of 13-cis-retinoic acid to all-trans-retinoic acid and the subsequent conversion to all-trans-retinoyl glucuronide are central events in the in vivo metabolism of 13-cis-retinoic acid in the rat. Moreover, the all-trans-retinoic acid detected in vivo could account for a significant fraction of the physiological activity of 13-cis-retinoic acid. The tissue disposition and metabolism of orally dosed 13-cis-[3H]retinoic acid are modulated by retinoid treatment. Chronic 13-cis-retinoic acid treatment apparently increased the intestinal accumulation of all-trans-retinoic acid, all-trans-retinoyl glucuronide, and 13-cis-retinoyl glucuronide. The liver concentrations of tritiated all-trans-retinoic acid and all-trans-retinoyl glucuronide were also elevated in 13-cis-retinoic acid treated rats.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Automated solid-phase extraction and liquid chromatographic method for retinoid determination in biological samples

In this study, a method for partly automated sample preparation and fully automated solid-phase extraction method for plasma, kidney and liver samples for various retinoids like all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, retinol and retinyl palmitate was established. Plasma, embryo-, kidney-and liver-homogenates were automatically mixed and extracted on multiple usage solid-phase (C2) extraction cartridges immediately before HPLC analysis. Automated cleaning, preconditioning and incorporation of the loaded cartridge to fully automated HPLC separation and quantification of the various retinoids in a single HPLC run was established. The recovery of the retinoids was generally between 80 and 90%. Intra-day repeatability was < 11.7%. As little as 1.2 ng/ml could be quantified in lipid-mixture standard samples. This method allows a highly automated sample preparation and a fully automated solid-phase extraction with good selectivity for the study of endogenous retinoids and retinoids after nutritional supplementations and pharmacological applications in several biological samples.