Vitamin D and calcium insufficiency-related chronic diseases: molecular and cellular pathophysiology.

A compromised vitamin D status, characterized by low 25-hydroxyvitamin D (25-(OH)D) serum levels, and a nutritional calcium deficit are widely encountered in European and North American countries, independent of age or gender. Both conditions are linked to the pathogenesis of many degenerative, malignant, inflammatory and metabolic diseases. Studies on tissue-specific expression and activity of vitamin D metabolizing enzymes, 25-(OH)D-1 alpha-hydroxylase and 25-(OH)D-24-hydroxylase, and of the extracellular calcium-sensing receptor (CaR) have led to the understanding of how, in non-renal tissues and cellular systems, locally produced 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and extracellular Ca(2+) act jointly as key regulators of cellular proliferation, differentiation and function. Impairment of cooperative signalling from the 1,25-(OH)(2)D(3)-activated vitamin D receptor (VDR) and from the CaR in vitamin D and calcium insufficiency causes cellular dysfunction in many organs and biological systems, and, therefore, increases the risk of diseases, particularly of osteoporosis, colorectal and breast cancer, inflammatory bowel disease, insulin-dependent diabetes mellitus type I, metabolic syndrome, diabetes mellitus type II, hypertension and cardiovascular disease. Understanding the underlying molecular and cellular processes provides a rationale for advocating adequate intake of vitamin D and calcium in all populations, thereby preventing many chronic diseases worldwide.

Vitamin D and calcium deficits predispose for multiple chronic diseases.

There is evidence from both observational studies and clinical trials that calcium malnutrition and hypovitaminosis D are predisposing conditions for various common chronic diseases. In addition to skeletal disorders, calcium and vitamin D deficits increase the risk of malignancies, particularly of colon, breast and prostate gland, of chronic inflammatory and autoimmune diseases (e.g. insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis), as well as of metabolic disorders (metabolic syndrome, hypertension). The aim of the present review was to provide improved understanding of the molecular and cellular processes by which deficits in calcium and vitamin D cause specific changes in cell and organ functions and thereby increase the risk for chronic diseases of different aetiology. 1,25-Dihydroxyvitamin D(3) and extracellular Ca(++) are both key regulators of proliferation, differentiation and function at the cellular level. However, the efficiency of vitamin D receptor-mediated intracellular signalling is limited by the negative effects of hypovitaminosis D on extrarenal 25-hydroxyvitamin D-1alpha-hydroxylase activity and thus on the production of 1,25-dihydroxyvitamin D(3). Calcium malnutrition eventually causes a decrease in calcium concentration in extracellular fluid compartments, resulting in organ-specific modulation of calcium-sensing receptor activity. Hence, attenuation of signal transduction from the ligand-activated vitamin D receptor and calcium-sensing receptor seems to be the prime mechanism by which calcium and vitamin D insufficiencies cause perturbation of cellular functions in bone, kidney, intestine, mammary and prostate glands, endocrine pancreas, vascular endothelium, and, importantly, in the immune system. The wide range of diseases associated with deficits in calcium and vitamin D in combination with the high prevalence of these conditions represents a special challenge for preventive medicine.

Assessment of vitamin D and calcium status in healthy adult Austrians.

BACKGROUND: Because there is reason to assume that also in Austria calcium and vitamin D malnutrition is wide-spread, we initiated a comprehensive study on calcium and vitamin D status in relation to bone health in a large group of the normal adult population. SUBJECTS AND METHODS: We assessed dietary calcium and vitamin D intake, serum concentrations of Ca2+, phosphate, alkaline phosphatase, 25(OH)D, 1,25(OH)2D, parathyroid hormone (PTH), follicle-stimulating hormone (FSH), sex hormones and bone mineral density (BMD) by double-energy X-ray absorptiometry at five different skeletal sites in 648 females and 400 males (age 21-76 years). RESULTS: Mean daily intake of vitamin D (101 IU, range 0.2-320) and calcium (569 mg, range 40-2170) was significantly less than the respective recommended dietary allowances. Two hundred and seventy-one (26%) individuals had hypovitaminosis D with serum 25(OH)D < 12 ng mL(-1), while serum Ca2+ was less than normal in 82 (7.8%) subjects. Multiple regression analysis revealed significant correlations between mean calcium intake and BMD in the femoral region in the men (r = 0.13, P < 0.05) though not in the women. No consistent data could be obtained for associations between BMD and vitamin D status, except for 25(OH)D and BMD at the spine in the men (r = 0.10, P < 0.05). 25(OH)D correlated negatively (P < 0.05) with age in the women (r = -0.11) and with PTH in the women (r = -0.11) and men (r = -0.16). Inversely, a significant (P < 0.001) age-related increase in PTH was observed in both sexes (men, r = 0.19; women, r = 0.14). CONCLUSIONS: Prevalence of hypovitaminosis D in adult Austrians is an imminent risk for development of secondary hyperparathyroidism with advancing age, and requires timely correction of nutritional deficits.

Normative data of bone mineral density in an unselected adult Austrian population.

BACKGROUND: There is increasing evidence that correct interpretation of bone mineral density (BMD) measurements by dual energy X-ray absorptiometry (DEXA) requires a population-specific reference range. We therefore collected data on age-related BMD in a random sample of the normal adult Austrian population to establish an appropriate normative database. METHODS: We measured BMD by DEXA at five different skeletal sites in 1089 subjects, i.e. 654 females and 435 males, aged between 21-76 years, who had been recruited by 17 centres across Austria. RESULTS: Age-related bone loss was observed until age 65 years with significant changes at the lumbar spine (r = -0.23), total hip (r = -0.07), trochanter (r = -0.10), femoral neck (r = -0.30) and Ward's triangle (r = -0.40) in the women but only at the femoral neck (r = -0.23) and at Ward's triangle (r = -0.40) in the men. When we calculated T scores from the BMD data of the young normal adult study population and used the T score set points according to the WHO classification of osteopenia and osteoporosis, we found that, depending on the skeletal site measured, 7.6-27.4% of the women and 16-41% of the men in our study group had low bone mass, whereas 0.6-2.7% of the female and 0.2-1.0% of the male study population were osteoporotic. However, osteoporosis was indicated in 4-9-fold more females and 5-15-fold more males when we based our estimates on the normative data provided by the manufacturers of the DEXA systems. CONCLUSION: Our data underscore the importance of using a population-specific reference range for DEXA measurements to avoid overdiagnosis of osteoporosis.

Quantitative ultrasound in the assessment of bone status of patients suffering from rheumatic diseases.

OBJECTIVE: To explore the effects of rheumatic diseases and glucocorticoids on bone mass a group of patients suffering from systemic lupus erythematosus (SLE, n=18) and rheumatoid arthritis (RA, n=22) were examined. DESIGN: We examined 40 patients and 48 controls with quantitative ultrasound (QUS) and dual-energy X-ray absorptiometry (DXA). RESULTS: QUS (broadband ultrasound attenuation, BUA; speed of sound, SOS) values were found to be significantly lower in patients than in controls ( P<0.001). QUS measurements were moderately correlated with DXA measurements (kappa score ( kappa) 0.28 at the lumbar spine, and 0.46 at the femoral neck). There were no significant relations between the dosage of glucocorticoids and QUS parameters. CONCLUSION: In patients suffering from inflammatory rheumatic diseases QUS values were significantly decreased. SOS but not BUA and DXA measurements reflected disease activity assessed by erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). QUS reflects different aspects of bone status compared with DXA.

Immune phenotype and intracellular cytokine production of peripheral blood mononuclear cells from postmenopausal patients with osteoporotic fractures.

A number of factors with known effects on bone turnover are also immune regulatory factors. Disturbances of bone remodeling thus may be a consequence of altered local immune reactivity. We therefore determined surface markers and intracellular cytokine production of peripheral blood mononuclear cells by four-color flow cytometry in 19 postmenopausal patients with established osteoporosis and a control group of 11 postmenopausal women without fragility fractures. No significant differences in bone mineral density as assessed by dual energy X-ray absorptiometry were observed between the two groups. The following surface markers and cytokines were studied: CD3, CD4, CD8, CD16, CD19, CD29, CD45RA, CD56, CD57, HLA-DR, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-13, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and granulocyte macrophage colony stimulating factor. In the fracture patients, the percentage of CD8+ cells co-expressing CD57 was increased (14+/-2 vs. 8+/-1%; p=0.03). Moreover, the proportion of CD8+ cells co-expressing TNF-alpha (47+/-5 vs. 33+/-4; p=0.05) and both TNF-alpha and IFN-gamma was significantly higher in the patients than the controls (41+/-6 vs. 22+/-3%; p=0.04). IL-1beta expression tended to be increased in monocytes from patients with established osteoporosis. Distinct subsets of CD8+ cells thus appear to contribute to the development of osteoporotic fractures.

25-hydroxy-vitamin d metabolism in human colon cancer cells during tumor progression.

RT-PCR analysis showed elevated expression of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) and of 25-hydroxyvitamin D-24-hydroxylase (24-OHase) in well differentiated human colon carcinomas in comparison to normal mucosa. Further tumor progression is associated with a rise in 1alpha-OHase but with no significant change in 24-OHase mRNA expression. Accordingly, HPLC analysis of 25-hydroxy-vitamin D3 metabolism in freshly isolated tumor cells indicated that well to moderately differentiated colon cancers in situ are able to produce 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) and convert it through 24-OHase activity into side-chain modified metabolites, 1,24,25-(OH)3-D3 and 1,25-(OH)2- 24-oxo-D3. Likewise, 25-(OH)-D3 is metabolized into 24,25-(OH)2D3, 23,25-(OH)2D3, and 23,25-(OH)2-24-oxo-D3. Poorly-differentiated cancers expressed low levels of 1alpha-OHase mRNA, whereas 24-OHase was even over-expressed. RT-PCR and HPLC analysis of vitamin D metabolism in primary culture cell clones strongly suggested that the extent of endogenously produced 1alpha,25-(OH)2-D3 was inversely related to 24-OHase activity, which could thus limit the antimitotic efficacy of 1alpha,25-(OH)2-D3 particularly at late stages of colon cancer progression.

Bone turnover markers and sex hormones in men with idiopathic osteoporosis.

BACKGROUND: In contrast to osteoporosis in postmenopausal women, osteoporosis in men has received much less attention. PATIENTS AND METHODS: We determined various biochemical parameters of bone metabolism and sex hormones in 31 men with idiopathic osteoporosis and 35 age matched control subjects. RESULTS: In the men with osteoporosis, a significantly increased urinary excretion of deoxypyridinoline (5.3 +/- 0.2 vs. 4.6 +/- 0.2 nmol mmol-1 creatinine; P = 0.033) in addition to increased serum levels of the c-terminal telopeptide of type I collagen (2677 +/- 230 vs. 2058 +/- 153 pmol; P = 0.037) were found. While parameters of bone formation were not significantly different in the patients and controls, serum bone sialoprotein levels were significantly decreased in the patients (3.7 +/- 0.8 vs. 12.4 +/- 4.0 ng mL-1; P = 0.021). Moreover, in men with idiopathic osteoporosis, lower levels of estradiol (91.3 +/- 5.8 vs. 114.6 +/- 7.8 pmol L-1; P = 0.044), higher levels of sex hormone binding globulin (31.5 +/- 3.1 vs. 24.2 +/- 1.4 nmol L-1; P = 0.034) and a decreased free androgen index (42.6 +/- 5.2 vs. 56.4 +/- 5.9; P = 0.016) were seen. Serum estradiol levels correlated negatively with several parameters of bone resorption. CONCLUSIONS: In men with idiopathic osteoporosis, bone resorption is increased and exceeds bone formation. The excessive bone resorption seen in idiopathic male osteoporosis may be due to decreased estradiol levels and low levels of bioavailable testosterone.

Phenotypic characteristics of human monocytes undergoing transendothelial migration.

In our study we characterised the immunophenotype of monocytes that migrated through an endothelial cell (EC) monolayer in vitro. We found that monocyte migration led to an enhanced expression of CD11a, CD33, CD45RO, CD54 [intercellular cell-adhesion molecule (ICAM)-1] and human leucocyte antigen-DR. The most striking increase was observed for ICAM-1 when ECs were activated with tumour necrosis factor-alpha and interleukin-1alpha. The results of our study indicate the following: (1) there is a characteristic immunophenotype on the surface of monocytes after transendothelial migration; (2) this phenotype seems to be induced by interactions between monocytes and ECs; and (3) this change is enhanced by the pretreatment of ECs with cytokines. Taken together, the results suggest that local cytokine production activating ECs is sufficient to enhance monocyte migration and that this, in turn, can induce changes consistent with an activated phenotype known to be interactive between antigen-presenting cells and T cells. These results have implications for our pathogenetic insights into rheumatoid arthritis.

25-Hydroxyvitamin D(3)-1alpha-hydroxylase and vitamin D receptor gene expression in human colonic mucosa is elevated during early cancerogenesis.

Human colorectal cancer cells not only express the nuclear vitamin D receptor (VDR) but are also endowed with 25-hydroxy-vitamin D(3)-1alpha-hydroxylase activity and therefore are able to produce the specific ligand for the VDR, the hormonally active steroid 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). In the present study we show by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) as well as by Western blotting and immunohistochemical methods, that in human large intestinal carcinomas expression of the genes encoding the 25-(OH)D(3)-1alpha-hydroxylase as well as the VDR increases in parallel with ongoing dedifferentiation in the early phase of cancerogenesis, whereas in poorly differentiated late stage carcinomas only low levels of the respective mRNAs can be detected. This indicates that, through up-regulation of this intrinsic 1alpha,25(OH)(2)D(3)/VDR system which mediates the anti-mitotic effects of the steroid hormone, colorectal cancer cells are apparently able to increase their potential for an autocrine counter-regulatory response to neoplastic cell growth, particularly in the early stages of malignancy.

Stimulatory effects of cartilage-derived morphogenetic proteins 1 and 2 on osteogenic differentiation of bone marrow stromal cells.

Cartilage-derived morphogenetic proteins 1 and 2 (CDMP-1 and CDMP-2) are members of the bone morphogenetic protein (BMP) family which play an important role in embryonic skeletal development. Throughout adult life, bone marrow-derived precursor cells maintain their ability to differentiate into osteoblasts in response to local growth factors. This study examines the osteogenic potential of CDMP-1, CDMP-2, BMP-6 and osteogenic protein 1 (OP-1) in bone marrow stromal cells (BMSC) and investigates the endogenous expression of CDMPs/BMPs and their respective activin receptor-like kinase (ALK) receptors. A 4-day exposure of BMSC to CDMP-1, CDMP-2, BMP-6, and OP-1 under serum-free conditions stimulated the progression of the osteogenic lineage in a dose-dependent manner as evaluated by alkaline phosphatase activity and osteocalcin synthesis. In contrast to the BMPs, CDMP-1 and especially CDMP-2 were significantly less osteogenic, as confirmed by Northern blot analysis. Moreover, BMSC were shown to express endogenously CDMP-2, BMP-2 to -6 and ALK-1, -2, -3, -5 and -6. Phenotypic characterization of BMSC by RT-PCR showed transcripts of the fat marker adipsin and the prechondrocytic marker procollagen type IIA; however, we were unable to detect the mature cartilage markers, procollagen type IIB and aggrecan, even after growth factor treatment. Our data indicate that CDMP-1, CDMP-2, BMP-6 and OP-1 enhance the osteogenic phenotype in BMSC, with CDMPs being clearly less osteogenic than BMPs. The endogenous expression of a variety of CDMPs/BMPs and their respective ALK receptors, suggests a possible involvement of these growth factors in the osteogenic differentiation of bone marrow progenitor cells.

Dietary calcium and growth modulation of human colon cancer cells: role of the extracellular calcium-sensing receptor.

Using the human colon adenocarcinoma-derived cell line Caco-2, we investigated the possible role of the Ca2+-sensing receptor (CaR) in mediating effects of extracellular Ca2+ on cellular proliferation. Caco-2 cells respond to low ambient [Ca2+]o by activation of the protein kinase C-signaling pathway, leading to upregulation of c-myc mRNA expression and thereby, finally, to alleviation from the G1/S phase control of the cell cycle. This proliferative response can be reverted by activation of the CaR either through raising [Ca2+]o or, respectively, by using the CaR agonist Gd3+ as a substitute for Ca2+. The inhibitory effect of [Ca2+]o on cell replication exhibits saturation kinetics (IC50 = 0.045 mM), indicating the existence of a highly sensitive CaR operating at low ambient [Ca2+]o. Specific immunostaining revealed the presence of CaR-positive cells in the crypt epithelium of normal human colonic mucosa as well as in glandular (i.e., differentiated structures) of carcinomatous lesions. This could provide a rationale for use of calcium supplements for intervention in early phases of colon tumorigenesis.

Immunocytochemical localization of the extracellular calcium-sensing receptor in normal and malignant human large intestinal mucosa.

We identified the parathyroid type Ca(2+)-sensing receptor (CaR) in normal human colon mucosa and in cancerous lesions at the mRNA and protein level. Polymerase chain reaction produced an amplification product from reverse-transcribed large intestinal RNA which corresponded in size and length to a 537-bp sequence from exon 7 of the CaR gene. With a specific antiserum against its extracellular domain, the CaR could be detected by immunostaining in normal human colon mucosa in cells preferentially located at the crypt base. The CaR protein was also expressed in tumors of the large bowel in all 20 patients examined. However, the great majority of CaR-positive cells in the adenocarcinomas inspected were confined to more differentiated areas exhibiting glandular-tubular structures. Poorly or undifferentiated regions were either devoid of specific immunoreactivity or contained only isolated CaR-positive cells. In the normal mucosa and in glandular-tubular structures of cancerous lesions, the CaR was exclusively expressed in chromogranin A-positive enteroendocrine cells and in only a small fraction of PCNA-positive cells.

Differential stimulation by PGE(2) and calcemic hormones of IL-6 in stromal/osteoblastic cells.

Formation of osteoclast-like cells in mouse bone marrow cultures induced by either 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), parathyroid hormone (PTH) or prostaglandin E(2) (PGE(2)), respectively, shows partial dependence on interleukin-6 receptor (IL-6R) activation. This suggests that locally produced IL-6 could be relevant for osteoclast formation. Therefore, we evaluated the effects of 1,25-(OH)(2)D(3), PTH, and PGE(2) on IL-6 production in stromal/osteoblastic cell lines. It appeared that these bone resorptive factors differed widely in their ability to modulate IL-6 mRNA expression and, consequently, protein synthesis in each of the cell lines studied. While 1,25-(OH)(2)D(3) was marginally effective only in ST2 cells, and PTH caused a 2- to 20-fold increase in IL-6 levels MC3T3-E1 and UMR-106 cells, PGE(2) enhanced IL-6 production in the ST2 and MC3T3-E1 cell line by two to three orders of magnitude, respectively, and also induced IL-6 in fibroblastic L929 cells. PGE(2)-stimulated IL-6 release from mesenchymal cells seems to be important for autocrine/paracrine control of osteoclast formation in health and disease.

Surface markers and transendothelial migration of dendritic cells from elderly subjects.

Age-related changes of immune functions have been extensively investigated in both humans and animal models; nevertheless, the literature on potential alterations of dendritic cells, potent antigen presenting cells responsible for initiating immune responses, with aging is very scarce. We studied the immuno-phenotype of peripheral blood dendritic cells of elderly and young subjects by three-color flow cytometry. In addition, the capacity of transendothelial migration, an important step in inflammatory reactions, of peripheral blood dendritic cells of elderly subjects was investigated in an in vitro model. The expression of HLA-DR in the peripheral blood dendritic cells of the elderly subjects was significantly decreased when compared to the young control subjects. The expression of various other surface markers was similar in the young and elderly subjects. The ability of transendothelial migration of dendritic cells was found to be unimpaired in the elderly subjects. Both in the young and elderly subjects a significantly higher expression of CD29, CD86, HLA-DR, and HLA-DQ in the dendritic cells that had migrated through the endothelium in comparison to nonadherent, nonmigrating cells was found. In the migrating dendritic cells of the elderly subjects a significantly increased expression of CD11c was observed, whereas the expression of CD54 was significantly enhanced in the migrating dendritic cells of the young subjects only. In conclusion, our results demonstrate intact functions and a normal immunophenotype of dendritic cells derived from elderly subjects. Dendritic cells thus seem to be functional and therefore are not responsible for the well-known decline of T cell functions with aging.

In situ mRNA hybridization analysis and immunolocalization of the vitamin D receptor in normal and carcinomatous human colonic mucosa: relation to epidermal growth factor receptor expression.

There is evidence that vitamin D receptor (VDR)-mediated action of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) could limit colon cancer cell growth particularly when induced by activation of the epidermal growth factor receptor (EGFR). We therefore wanted to ascertain the relevance of this observation for human colon cancerogenesis. Utilizing in situ mRNA hybridization and immunocytochemical techniques, we analyzed cell-specific expression of VDR and EGFR in normal and malignant human colonic mucosa. In normal mucosa, VDR positivity is weak and observed only in a small number of luminal surface colonocytes. In contrast, EGFR expression at a relatively high level is also found in cells at the crypt base. The number of VDR-positive colonocytes increases remarkably during tumor progression. It reaches its maximum in low grade adenocarcinomas and returns to lower levels in highly malignant cancers. In both low- and high grade carcinomas, the great majority of tumor cells contain the EGFR message. The relative abundance of EGFR over VDR in normal mucosa and in high grade carcinomas would create a situation in which mitogenic effects from EGFR activation are only ineffectively counteracted by signaling from 1 alpha,25-(OH)2D3/VDR. In contrast, in well to moderately differentiated tumors, upregulation of VDR could retard further tumor progression.

Regulatory effects of 1alpha,25-dihydroxyvitamin D3 on the cytokine production of human peripheral blood lymphocytes.

We studied the possible regulatory effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] on cytokine production and differentiation of subsets of CD4+ [T helper 1 (Th1) and Th2] and CD8+ [T cytotoxic 1 (Tc1) and Tc2] lymphocytes at the single cell level. PBMC from healthy donors were cultured with or without 1alpha,25-(OH)2D3 for up to 21 days. On days 0, 7, 14, and 21, the percentage of cytokine-producing T lymphocytes was analyzed by intracellular cytokine detection with mAb and flow cytometry. Simultaneous staining for cell surface markers allowed discrimination of CD4+ and CD8+ T cell subsets. After culture with 1alpha,25-(OH)2D3 (10(-8) mol/L), no significant effects on the proportion of interferon-gamma (IFNgamma)- or interleukin-4 (IL-4)-producing cells were detected, whereas reduced frequencies of IL-2-producing cells in the CD4+ as well as in the CD8+ population were found. An increase in the low percentage of CD4+ and CD8+ T cells producing the Th2 cytokine IL-13 was noticed. Most interestingly, IL-6-producing CD4+ and CD8+ T cells could only be detected in cultures with 1alpha,25-(OH)2D3, reaching a plateau after 14 days. The percentage of IL-6-producing T cells induced by 1alpha,25-(OH)2D3 after a given time period remained stable for at least 7 weeks. Studies of cytokine coexpression revealed that about 70% of IL-6-producing CD4+ and CD8+ cells were also positive for IL-2, but more than 90% were negative for IFNgamma, IL-4, or IL-13, respectively. This suggests that the IL-6-producing population does not match the Th1/Tc1-like (IFNgamma+) or Th2/Tc2-like (IL-4+ or IL-13+) subset. The influence of 1alpha,25-(OH)2D3 on cytokine production by lymphocytes is probably an important point of intersection between the endocrine and the immune system.

Biological effects of 1alpha-hydroxy- and 1beta-(hydroxymethyl)-vitamin D compounds relevant for potential colorectal cancer therapy.

1alpha,25-dihydroxyvitamin D(3) and two synthetic analogs, 1alpha, 25-dihydroxy-16-ene-23-yne-vitamin D(3) (Ro 23-7553) and 1alpha, 25-dihydroxy-16-ene-24-oxo-vitamin D(3) (JK-1624-3), were tested for their ability to specifically inhibit growth and promote differentiation of human colon cancer cells in comparison with a series of 1beta-(hydroxymethyl) congeners of the natural hormone, such as 1beta-(hydroxymethyl)-3alpha,25(OH)(2)-16-ene,24-oxo-vitamin D(3) (JK-1624-2), 1beta-(hydroxymethyl)-3alpha, 25-dihydroxy-16-ene-26,27-dihomo vitamin D(3) (JK-1626-2), and 1beta-(hydroxymethyl)-3alpha,25-dihydroxy-22,24-diene-26,27- dihomo vitamin D(3) (MCW-EE). Western blot analysis revealed that reduction of cyclin D1 levels is a key mechanism by which the vitamin D compounds under investigation inhibit Caco-2 tumor cell growth. Both the 1alpha-hydroxy- as well as the 1beta-hydroxymethyl-type vitamin D compounds, which exhibit only low affinity for the vitamin D receptor, significantly reduced [(3)H]thymidine DNA labeling in confluent Caco-2 cell cultures. This suggests that high-affinity binding to the vitamin D receptor is not an absolute prerequisite for genomic action on tumor cell growth. Hybrid analogs JK-1624-2 and MCW-EE, although antimitotically active, were rather ineffective in promoting phenotypic differentiation of human colon cancer cells. However, because both compounds also do not promote osteoclast differentiation from hematopoetic bone marrow cells, they still could be used as antimitotic agents in cancer therapy, even at dose levels that, with other analogs, could cause hypercalcemia.

Mechanism of antimitogenic action of vitamin D in human colon carcinoma cells: relevance for suppression of epidermal growth factor-stimulated cell growth.

Because the efficacy of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] in treatment of colon cancer might critically depend on its ability to specifically counteract epidermal growth factor (EGF)-stimulated tumor cell growth, we utilized human colon adenocarcinoma-derived cells in primary culture as well as the Caco-2 cell line to elucidate possible sites of interaction of 1alpha,25-(OH)2D3 with signaling from EGF receptor activation. In both types of colon cancer cells investigated, 10(-8) M 1alpha,25-(OH)2D3 reduced basal cell proliferation by about 50%, and prevented any rise in proliferation when colon cancer cells were treated with 25 ng/ml EGF: this can be explained by a marked inhibitory effect of 1alpha,25-(OH)2D3 on EGFR mRNA and protein expression. The steroid hormone also seemingly promotes EGF-induced internalization of apical and basolateral membrane EGFR. In addition, 1alpha,25-(OH)2D3 significantly reduced basal and EGF-stimulated expression of cyclin D1 at the mRNA and protein level in primary cultures as well as in the Caco-2 cell line. The ability of 1alpha,25-(OH)2D3 to interfere with a key event in cell cycle control and thereby to block mitogenic signaling from EGF could be seen as advantageous for the potential use of vitamin D compounds in colon cancer therapy.

Expression of the vitamin D receptor, of estrogen and thyroid hormone receptor alpha- and beta-isoforms, and of the androgen receptor in cultures of native mouse bone marrow and of stromal/osteoblastic cells.

Marrow stromal cells mediate the effect of 1alpha,25-dihydroxyvitamin D3 on formation of osteoclast-like cells from undifferentiated hematopoetic precursors in bone marrow. Induction by the vitamin D hormone of multinucleated, calcitonin receptor- and tartrate-resistant acid phosphatase-positive cells in primary mouse bone marrow culture can be modulated by other members of the steroid/thyroid hormone family, such as triiodothyronine, which has a positive effect, as well as 17beta-estradiol and 5alpha-dihydrotestosterone, which both act as inhibitors of osteoclastogenesis. In an attempt to relate these effects of the steroid/thyroid hormones to the presence of their respective nuclear receptors, we studied expression of the vitamin D receptor (VDR), estrogen receptor (ER)-alpha and -beta, thyroid hormone receptor (TR)-alpha and -beta, and androgen receptor (AR) in total bone marrow as well as primary marrow stromal cell cultures. By using reverse-transcriptase-polymerase chain reaction, in both cases amplification products were obtained, which were identified by multiple restriction fragment length analysis as transcripts from mRNA specific for the ligand-binding domains of the VDR, ER-alpha, ER-beta, TR-alpha, TR-beta, and AR. Specific immunostaining by indirect peroxidase labeling revealed that among the various cell types present in bone marrow, the steroid/ thyroid hormone receptors are abundant particularly in marrow stromal cells. In another series of experiments, we extended our survey on receptor expression also to stromal/osteoblastic cell lines. At the mRNA level, the complete repertoire of steroid/thyroid hormone receptors was present in preadipocytic ST2 cells as well as in osteoblastic MC3T3-E1 cells. By immunocytochemical staining of the latter, it became apparent that single cells exhibit wide variations in intensity of specific signals for all the receptors investigated, so that, notably in contrast to primary stromal cells and ST2 cells, MC3T3-E1 display a mosaic pattern of receptor protein expression.