Vitamin D and skin cancer: a problem in gene regulation

The skin is the major source of Vitamin D(3) (cholecalciferol), and ultraviolet light (UV) is critical for its formation. Keratinocytes, the major cell in the epidermis, can further convert Vitamin D(3) to its hormonal form, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] (calcitriol). 1,25(OH)(2)D(3) in turn stimulates the differentiation of keratinocytes, raising the hope that 1,25(OH)(2)D(3) may prevent the development of malignancies in these cells. Skin cancers (squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanomas) are the most common cancers afflicting humans. UV exposure is linked to the incidence of these cancers-UV is thus good and bad for epidermal health. Our focus is on the mechanisms by which 1,25(OH)(2)D(3) regulates the differentiation of keratinocytes, and how this regulation breaks down in transformed cells. Skin cancers produce 1,25(OH)(2)D(3), contain ample amounts of the Vitamin D receptor (VDR), and respond to 1,25(OH)(2)D(3) with respect to induction of the 24-hydroxylase, but fail to differentiate in response to 1,25(OH)(2)D(3). Why not? The explanation may lie in the overexpression of the DRIP complex, which by interfering with the normal transition from DRIP to SRC as coactivators of the VDR during differentiation, block the induction of genes required for 1,25(OH)(2)D(3)-induced differentiation.     

1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is an immunomodulatory agent inducing dendritic cells (DCs) to become tolerogenic. To further understand its mechanisms of action, we have examined the effects of 1,25(OH)(2)D(3) on tolerogenic properties of blood myeloid (M-DCs) and plasmacytoid (P-DCs) human DC subsets. Exposure of M-DCs to 1,25(OH)(2)D(3) up-regulated production of CCL22, a chemokine attracting regulatory T cells, whereas production of CCL17, the other CCR4 ligand, was reduced. 1,25(OH)(2)D(3) also decreased IL-12p75 production by M-DCs, as expected, and inhibited CCR7 expression. 1,25(OH)(2)D(3) treatment markedly increased CD4(+) suppressor T cell activity while decreasing the capacity of M-DCs to induce Th1 cell development. Surprisingly, 1,25(OH)(2)D(3) did not exert any discernible effect on tolerogenic properties of P-DCs, and even their high production of IFN-alpha was not modulated. In particular, the intrinsically high capacity of P-DCs to induce CD4(+) suppressor T cells was unaffected by 1,25(OH)(2)D(3). Both DC subsets expressed similar levels of the vitamin D receptor, and its ligation by 1,25(OH)(2)D(3) similarly activated the primary response gene cyp24. Interestingly, 1,25(OH)(2)D(3) inhibited NF-kappaB p65 phosphorylation and nuclear translocation in M-DCs but not P-DCs, suggesting a mechanism for the inability of 1,25(OH)(2)D(3) to modulate tolerogenic properties in P-DCs.     

NOD bone marrow-derived dendritic cells are modulated by analogs of 1,25-dihydroxyvitamin D3

The immune effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mainly mediated through dendritic cells (DCs). In vitro, 1,25(OH)(2)D(3) treatment renders murine bone marrow (BM)-derived DCs more tolerogenic, indirectly altering behavior and fate of T lymphocytes. In vivo, treatment with 1,25(OH)(2)D(3) or its analogs prevents diabetes in NOD mice. The aim of this study was to investigate the effects of the 1,25(OH)(2)D(3)-analog TX527 on the expression of antigen-presenting and costimulatory/migratory molecules on BM-derived DCs from NOD mice. After culture with 20 ng/ml GM-CSF + 20 ng/ml IL-4 (8 days) followed by 1000 ng/ml LPS + 100 U/ml IFN-gamma (2 days), with or without 10(-8)M TX527, cells were counted and analyzed by FACS for MHC II, CD86, CD40 and CD54 expression within the CD11c(+) DC population. Upon TX527 treatment, cell recovery was significantly reduced whereas the CD11c(+) DC fraction remained constant. On CD11c(+) DCs, MHC II, CD86 and CD54 were significantly down-regulated and CD40 was twofold upregulated. Globally, BM-derived DCs from NOD mice become more tolerogenic upon TX527 treatment, confirming the effects of 1,25(OH)(2)D(3) on murine DCs and possibly explaining the protective effects of 1,25(OH)(2)D(3) and its analogs from diabetes in NOD mice.     

Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)(2)D(3) levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)(2)D(3) on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)(2)D(3), although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1 alpha-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)(2)D(3) and/or activation. Importantly, 1,25(OH)(2)D(3) up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)(2)D(3) may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.     

Nuclear lipid microdomains regulate nuclear vitamin D3 uptake and influence embryonic hippocampal cell differentiation

Despite recent advances in the understanding of the role of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) in the CNS, the mechanism of action remains obscure. We demonstrate that some 1,25-(OH)(2)D(3) receptor (VDR) is localized in the cell nucleus in specialized microdomains enriched in sphingomyelin and cholesterol; the integrity of these microdomains is necessary for embryonic hippocampal cell differentiation. Sphingomyelinase (SMase) treatment reduces both VDR and labeled 1,25-(OH)(2)D(3) content in nuclear microdomains. We have previously shown that HN9.10e embryonic hippocampal cells differentiate when incubated with 100 nM 1,25-(OH)(2)D(3) in the presence of 10% fetal calf serum, while serum deprivation induces cell death. In this study, we have investigated whether conditions that alter lipid content of nuclear microdomains modify 1,25-(OH)(2)D(3)-induced differentiation. Serum deprivation activates SMase and modifies the composition of nuclear microdomains, which lose the 1,25-(OH)(2) vitamin D(3) receptor. The incubation of serum-deprived cells with 100 nM 1,25-(OH)(2)D(3) prevents differentiation. However, treatment with 400 nM 1,25-(OH)(2)D(3) during serum withdrawal increases the lipid content of the nuclear microdomains, allows the interaction of 1,25-(OH)(2)D(3) with its receptor, and results in differentiation. These results suggest the presence of VDR in nuclear microdomains is necessary for 1,25-(OH)(2)D(3)-induced differentiation in embryonic hippocampal cells.     

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.     

Transforming growth factor-beta 1 signaling contributes to Caco-2 cell growth inhibition induced by 1,25(OH)(2)D(3)

Growth of Caco-2 and many cancer cells is inhibited by 1,25(OH)(2)D(3). Whereas TGF-beta 1 inhibits normal colonic epithelial cell growth, most human colon cancer-derived cells, including Caco-2 and SW480 cells, are resistant to it. The mechanisms underlying these antiproliferative actions and resistance to TGF-beta growth inhibition are largely unknown. We observed that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] sensitized Caco-2 and SW480 cells to TGF-beta 1 growth inhibitory effects. Versus 1,25(OH)(2)D(3) alone, the combination of 1,25(OH)(2)D(3) and TGF-beta 1 significantly reduced cell numbers. Also, the amount of active TGF-beta 1 was increased (~4-fold) by this secosteroid in conditioned media from Caco-2 cells. The 1,25(OH)(2)D(3) increased the expression of IGF-II receptors (IGF-IIR), which facilitated activation of latent TGF-beta 1, and was found to activate TGF-beta signaling in Caco-2 cells. By using neutralizing antibodies to human TGF-beta 1, we showed that this cytokine contributes to secosteroid-induced inhibition of Caco-2 cell growth. Also, 1,25(OH)(2)D(3) was found to enhance the type I TGF-beta receptor mRNA and protein abundance in Caco-2 cells. Whereas the 1,25(OH)(2)D(3)-induced sensitization of Caco-2 cells to TGF-beta 1 was IGF-IIR independent, the type I TGF-beta 1 receptor was required for this sensitization. Thus 1,25(OH)(2)D(3) treatment of Caco-2 cells results in activation of latent TGF-beta 1, facilitated by the enhanced expression of IGF-IIR by this secosteroid. Also, 1,25(OH)(2)D(3) sensitized Caco-2 cells to growth inhibitory effects of TGF-beta 1, contributing to the inhibition of Caco-2 cell growth by this secosteroid.     

PTHrP contributes to the anti-proliferative and integrin alpha6beta4-regulating effects of 1,25-dihydroxyvitamin D(3)

Parathyroid hormone-related protein (PTHrP) increases the growth and metastatic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] suppresses PTHrP expression and exerts an anti-proliferative effect in prostate carcinoma cells. We used the human prostate cancer cell line C4-2 as a model system to ask whether down-regulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the anti-proliferative effects of 1,25(OH)(2)D(3). Since PTHrP increases the expression of the pro-invasive integrin alpha6beta4, we also asked whether 1,25(OH)(2)D(3) decreases integrin alpha6beta4 expression in C4-2 cells, and whether modulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the effects of 1,25(OH)(2)D(3) on integrin alpha6beta4 expression. Two strategies were utilized to modulate PTHrP levels: overexpression of PTHrP (-36 to +139) and suppression of endogenous PTHrP expression using siRNAs. We report a direct correlation between PTHrP expression, C4-2 cell proliferation and integrin alpha6beta4 expression at the mRNA and cell surface protein level. Treatment of parental C4-2 cells with 1,25(OH)(2)D(3) decreased cell proliferation and integrin alpha6 and beta4 expression. These 1,25(OH)(2)D(3) effects were significantly attenuated in cells with suppressed PTHrP expression. 1,25(OH)(2)D(3) regulates PTHrP expression via a negative vitamin D response element (nVDRE) within the noncoding region of the PTHrP gene. The effects of 1,25(OH)(2)D(3) on cell proliferation and integrin alpha6beta4 expression were significantly attenuated in cells overexpressing PTHrP (-36 to +139), which lacks the nVDRE. These findings suggest that one of the pathways via which 1,25(OH)(2)D(3) exerts its anti-proliferative effects is through down-regulation of PTHrP expression.     

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis,Jun kinase activation,and interleukin-6 production in primary human keratinocytes

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.     

Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome II.

Cytogenetic and molecular genetic analyses of human intraspecific HeLa x fibroblast hybrids have provided evidence for the presence of a tumor-suppressor gene(s) on chromosome 11 of normal cells. In the present study, we have carried out extensive RFLP analysis of various nontumorigenic and tumorigenic hybrids with at least 50 different chromosome 11-specific probes to determine the precise location of this tumor-suppressor gene(s). Two different hybrid systems, (1) microcell hybrids derived by the transfer of a normal chromosome 11 into a tumorigenic HeLa-derived hybrid cell and (2) somatic cell hybrids derived by the fusion of the HeLa (D98OR) cells to a retinoblastoma (Y79) cell line, were particularly informative. The analysis showed that all but one of the nontumorigenic hybrid cell lines contained a complete copy of the normal chromosome 11. This variant hybrid contained a segment of the long arm but had lost the entire short arm of the chromosome. The tumorigenic microcell and somatic cell hybrids had retained the short arm of the chromosome but had lost at least the q13-23 region of the chromosome. Thus, these results showed a perfect correlation between the presence of the long arm of chromosome 11 and the suppression of the tumorigenic phenotype. We conclude therefore that the gene(s) involved in the suppression of the HeLa cell tumors is localized to the long arm (q arm) of chromosome 11.