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Biochemical, Molecular, and Genetic Mechanisms

Human Mammary Epithelial Cells Express CYP27B1 and Are Growth Inhibited by 25-Hydroxyvitamin D-3, the Major Circulating Form of Vitamin D-3¹

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556

³ To whom correspondence should be addressed. E-mail: jwelsh3{at}nd.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
1,25-dihydroxycholecalciferol [1,25(OH)₂D₃], the active form of cholecalciferol, is a negative growth regulator of breast cancer cells. CYP27B1 is a cytochrome P450-containing hydroxylase expressed in kidney and other tissues that generates 1,25(OH)₂D₃ from an inactive vitamin D precursor 25-hydroxycholecalciferol [25(OH)D₃]. In these studies, we tested the hypothesis that mammary cells express CYP27B1 and locally produce 1,25(OH)₂D₃, which acts in an autocrine manner to regulate cell turnover. Using Western blot and quantitative real-time PCR, CYP27B1 mRNA and protein were detected in immortalized, nontumorigenic human mammary epithelial cell (HMEC) cultures. Furthermore, HMEC cultures were dose dependently growth inhibited by physiological concentrations of 25(OH)D₃, suggesting that CYP27B1 converts this precursor cholecalciferol metabolite to 1,25(OH)₂D₃, the ligand for the vitamin D receptor (VDR). In support of this suggestion, both 1,25(OH)₂D₃ and 25(OH)D₃ transactivated VDR in HMEC cultures, as measured by induction of a vitamin D responsive reporter gene and upregulation of CYP24, an endogenous VDR target gene. No induction of CYP24 by 25(OH)D₃ was observed in mammary cells derived from CYP27B1 null mice. Similar results were observed in 2 independently derived immortalized HMEC lines as well as in primary cultures derived from human breast epithelium. These are the first studies to demonstrate that nontransformed human mammary cells express CYP27B1, that they are growth inhibited by physiologically relevant concentrations of 25(OH)D₃, and that they provide a biological mechanism linking vitamin D status to breast cancer risk.

KEY WORDS: • vitamin D • mammary • breast cancer • CYP27B1

The vitamin D steroids ergocalciferol (D-2) and cholecalciferol (D-3) modulate calcium homeostasis, cell turnover, and immune responses in a variety of tissues. Vitamin D-2 and D-3 can be obtained from natural foods, fortified products, and supplements, and vitamin D-3 can be synthesized from 7-dehydrocholesterol in skin exposed to UVB radiation (sunlight). Regardless of source, vitamins D-2 and D-3 exert biological activity only after a series of hydroxylations catalyzed by cytochrome P450 (CYP)⁴-containing enzymes. The first of these conversions is catalyzed by CYP27A, a vitamin D 25-hydroxylase, which metabolizes cholecalciferol to 25-hydroxycholecalciferol [25(OH)D₃], a circulating metabolite present in the nmol/L range that correlates with vitamin D-3 status (1). Although 25(OH)D₃ is the major circulating form of cholecalciferol, its only known function is to serve as a precursor to 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃], the biologically active metabolite generated by CYP27B1 [25(OH)D₃ 1-hydroxylase], a mitochondrial enzyme present in renal proximal tubules (2). Renal CYP27B1 activity is inversely correlated with calcium status, and serum concentrations of 1,25(OH)₂D₃ are kept in the pmol/L range through classical negative feedback mechanisms. Thus, under conditions of normocalcemia, renal CYP27B1 activity is inhibited and 25(OH)D₃ is instead metabolized by CYP24 (a vitamin D 24-hydroxylase) to 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], a biologically inactive metabolite that is ultimately converted to calcitroic acid and excreted (3).

In addition to its role in calcium homeostasis, epidemiologic studies suggest that optimal vitamin D status has a protective effect against the formation and progression of several common cancers (4–6). 1,25(OH)₂D₃ interacts with the vitamin D receptor (VDR) to inhibit directly the growth of prostate, colon, and breast cancer cells (7–10); however, these growth regulatory effects are observed at concentrations (100 nmol/L) well above the physiologic range that are in fact toxic in vivo. Based on these considerations, it is unlikely that 1,25(OH)₂D₃ acts at the systemic level to regulate cell growth in vivo. The identification of CYP27B1 in skin, colon, prostate, and breast (11–15) suggests that locally generated 1,25(OH)₂D₃ could act in an autocrine manner to protect cells against transformation. In support of this concept, low circulating levels of the CYP27B1 substrate, 25(OH)D_3, are positively (1) correlated with biomarkers and/or risk for prostate, colon, and breast cancer (5,16–18). Moreover, extrarenal expression of CYP27B1 appears to be of biological significance because locally generated 1,25(OH)₂D₃ inhibited growth and induced differentiation of transformed keratinocytes in a xenograft model (19). In addition, loss of CYP27B1 in prostate cancer cells correlated with reduced sensitivity to 25(OH)D₃ (14).

The hypothesis that CYP27B1 in extrarenal tissues may generate sufficient 1,25(OH)₂D₃ to affect cell transformation predicts that normal epithelial cells would express both VDR and CYP27B1, a prediction that has already held true for normal keratinocytes, colonocytes, and prostate epithelial cells (12,14). Although CYP27B1 is expressed in human and murine mammary tissue and breast cancer cell lines (20,21), little is known about vitamin D metabolism in nontumorigenic mammary epithelial cells. In this study, we demonstrate that both immortalized and primary cells derived from human breast express CYP27B1, and that 25(OH)D₃ regulates expression of VDR target genes and inhibits cell growth at physiologic concentrations. These data suggest that the concentration of circulating 25(OH)D₃ may dictate the ability of mammary cells to synthesize 1,25(OH)₂D₃, which could affect cell turnover, thus providing a biological basis for the epidemiologic data linking vitamin D status to breast cancer risk.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Cell culture. Primary human mammary epithelial cells (PHMEC) and a telomerase-immortalized, nontumorigenic human mammary epithelial cell (HMEC) line were obtained from Cambrex. The data obtained in the HMEC cultures were confirmed in an independently derived telomerase-immortalized HMEC line donated by Dr. Robert Weinberg (MIT, Cambridge, MA) (22); however, for simplicity, only data from the commercially available cells are shown. Although capable of infinite population doublings in vitro, telomerase-immortalized HMEC cultures retain morphology and growth characteristics of normal mammary epithelial cells, and are not tumorigenic in the absence of additional genetic mutations (22). MCF-7 breast cancer cells were obtained from the American Type Culture Collection, and HKC-8 SV-40 immortalized human proximal kidney cells were provided by Dr. Lorraine Racusen (Johns Hopkins University, Baltimore, MD). All cell lines were grown at 37C° and 5% CO₂ in a humidified incubator. HMEC and PHMEC were cultured in Medium171 (Cascade Biologics) supplemented with 0.4% v:v bovine pituitary extract, 5 mg/L bovine insulin, 0.5 mg/L hydrocortisone, and 3 µg/L human epidermal growth factor (MEGS, Cascade Biologics). MCF-7 cells were cultured in MEM containing 5% fetal bovine serum. HKC-8 cells were cultured in DMEM/F-12 containing 5% fetal bovine serum. Primary mammary cells from CYP27B1 knockout mice (23) were isolated by collagenase digestion as described (8) and cultured in Medium171 supplemented with MEGS.

    Growth assay. Exponentially growing HMEC, PHMEC, and MCF-7 cultures were treated 1 d after plating with ethanol vehicle, 1,25(OH)₂D₃ or 25(OH)D₃, at the concentrations indicated in the figure legends. For time-course studies, cells were harvested before treatment and after 24, 48, 72, and 96 h with no media changes; for all other studies, media were replenished after 72 h. Cultures were fixed with 1% glutaraldehyde and stained with 0.1% crystal violet. After solubilization of the stain in 0.1% Triton X-100, absorbance was measured at 590 nm as an indicator of cell density.

    Immunoblotting. Subconfluent cultures of HMEC, PHMEC, MCF-7, HKC-8, and primary murine cells were treated with ethanol vehicle, 1,25(OH)₂D₃, or 25(OH)D₃ for 48 h. Monolayers were scraped into laemmli buffer containing protease and phosphatase inhibitors (10 mmol/L benzamidine, 10 mmol/L sodium fluoride, 100 mmol/L sodium vanadate, 25 µg/µL aprotinin, 25 µg/µL pepstatin, 25 µg/µL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride) and sonicated. After separation on 10% SDS-PAGE, transfer to nitrocellulose, and blocking with 20% skim milk, primary antibodies were applied at 1:40 (VDR Clone 9A7, NeoMarkers), 1:200 (CYP24, donated by Cytochroma) or 1:500 (CYP27B1, The Binding Site) dilutions. After being washed in PBS/0.1% Tween, horseradish peroxidase conjugated anti-rat (Santa Cruz Biotechnology), anti-mouse (Amersham Biosciences) or anti-sheep (Jackson ImmunoResearch) secondary antibodies were applied and VDR, CYP24, and CYP27B1 abundance was detected by chemiluminescence. Specific bands were detected at 52 kDa (VDR), 50 kDa (CYP24), and 56 kDa (CYP 27B1). Ponceau staining was used to confirm equal loading, and in some cases, blots were stripped and reprobed with anti-GAPDH.

    Quantitative real-time PCR. Subconfluent HMEC, MCF-7, and HKC-8 cells were treated with ethanol vehicle, 100 nmol/L 1,25(OH)₂D₃, or 100 nmol/L 25(OH)D₃ for 24h, and total RNA was extracted with RNeasy Mini Kit (Qiagen). cDNA synthesized with TaqMan Reverse Transcription Reagents (Applied Biosystems) was analyzed by real-time PCR using TaqMan PCR Core Reagent Kit (Applied Biosystems) and primers and probes specific for CYP27B1 (forward: AGTTGCTATTGGCGGGAGTG; reverse: GTGCCGGGAGAGCTCATACA, probe: ACTACCGCAAAGAAGGCTACGGGCTG) and CYP24 (forward: CAAACCGTGGAAGGCCTATC, reverse: AGTCTTCCCCTTCCAGGATCA, probe: ACTACCGCAAAGAAGGCTACGGGCTG). CYP27B1 and CYP24 expression was normalized against 18S RNA (forward: AGTCCCTGCCCTTTGTACACA, reverse: GATCCGAGGGCCTCACTAAAC, probe: CGCCCGTCGCTACTACCGATTGG) expression, which was analyzed in parallel.

    Transient transfections. Subconfluent HMEC, PHMEC, MCF-7, and HKC-8 cells were co-transfected in serum-free medium with a 300-bp vitamin D–responsive region of the CYP24 promoter driving the firefly luciferase reporter gene (pGL3–24OH, 0.64 µg, obtained from the late Dr. Jack Omdahl) and the thymidine kinase promoter driving the renilla luciferase reporter gene (pRL-TK, 0.16µg, Promega) using a 3:2 (v:wt) ratio of FUGENE:DNA. After 4 h, the cells were treated with ethanol vehicle, 100 nmol/L 1,25(OH)₂D₃, or 100 nmol/L 25(OH)D₃ in complete media conditions for 24h. Dual luciferase assays were performed using reagents from Promega, and pGL3-24OH values were normalized to pRL-TK. Data are presented as relative luciferase units (RLU) where control values were set to 1 for each cell line.

    Statistical analyses. Data are expressed as means ± SE, with the number of replicates indicated in each figure legend. Student's t test and 1-way ANOVA followed by Tukey's multiple comparison test were used [Instat Software (GraphPad)]. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    VDR is expressed and functional in non-transformed mammary cells. The growth inhibitory effects of 1,25(OH)₂D₃, the VDR ligand, in breast cancer cells are well recognized; however, little is known about vitamin D signaling in nontumorigenic mammary cells. Therefore, we compared VDR expression and function in HMEC, MCF-7, and HKC-8 cells (Fig, 1). VDR was detected in all cell lines, but was highest in the noncancerous HMEC cultures and lowest in HKC-8 renal cells (Fig. 1A). In all cell lines, 100 nmol/L 1,25(OH)₂D₃ significantly (P < 0.05) induced the vitamin D responsive reporter gene containing the CYP24 promoter (Fig. 1B). Similarly, 100 nmol/L 1,25(OH)₂D₃ significantly (P < 0.05) enhanced CYP24 mRNA and protein expression in HMEC, MCF-7 and HKC-8 cells (Fig. 1C,D). Based on the magnitude of CYP24 induction, MCF-7 cells were more sensitive to 1,25(OH)₂D₃ than HMEC and HKC-8 cultures. Collectively, these data indicate that the VDR is expressed and functional in HMEC cultures, and that this signaling pathway remains intact in MCF-7 breast cancer cells.

View larger version (27K): [in this window] [in a new window]   FIGURE 1  Expression and function of VDR in HMEC, MCF-7 and HKC-8 cells. (A) Lysates from HMEC, MCF-7, and HKC-8 cells were immunoblotted with an antibody against VDR. (B) HMEC, MCF-7, and HKC-8 cells were co-transfected with a CYP24 reporter gene and a normalization gene and treated for 24 h with vehicle or 100 nmol/L 1,25(OH)2D3. For each cell line, the RLU for vehicle-treated cells were set to 1 and fold induction by 1,25(OH)2D3 is shown. Values are means ± SEM, n = 4. (C) CYP24 mRNA expression in cells treated with vehicle or 100 nmol/L 1,25(OH)2D3 for 24 h was assessed by real-time PCR and normalized against 18S RNA. For each cell line, data are expressed as fold of the control cells. Values are means ± SEM, n = 3. (D) Lysates from cells treated with 100 nmol/L 1,25(OH)2D3 for 24 h were immunoblotted with antibodies against CYP24 (top panel) and GAPDH (bottom panel). *Different from the control, P < 0.01.

View larger version (12K): [in this window] [in a new window]   FIGURE 2  Effect of 1,25(OH)2D3 on growth of HMEC and MCF-7 cells. HMEC (upper panel) and MCF-7 (lower panel) cells were treated for 96 h with ethanol vehicle or increasing concentrations of 1,25(OH)2D3. Cell density was assessed by crystal violet assay and expressed as a percentage of the control. Values are means ± SEM, n = 3. Means not sharing a common letter differ, P < 0.05.

View larger version (19K): [in this window] [in a new window]   FIGURE 3  Comparative gene expression of CYP27B1 in HMEC, MCF-7, and HKC-8 cells. (A) CYP27B1 mRNA expression was determined by real-time PCR and expressed as relative gene expression after normalization to 18S RNA. Bars not sharing a common letter differ, P < 0.05. (B) Lysates from HMEC and MCF-7 cells were immunoblotted with an antibody against CYP27B1. (C) CYP27B1 mRNA expression in HMEC, MCF-7, and HKC-8 cultures treated with vehicle or 100 nmol/L 1,25(OH)2D3 for 24 h was assessed by real-time PCR as described in (A). For each cell line, data are expressed as fold of the control cells. Values are means ± SEM, n = 3. *Different from the control, P < 0.01.

View larger version (17K): [in this window] [in a new window]   FIGURE 4  Effect of 25(OH)D3 on growth and regulation of VDR target genes in mammary cell cultures. (A) Cell density of HMEC cultures treated for 96 h with ethanol vehicle or increasing concentrations of 25(OH)D3. Data were obtained by crystal violet staining and are expressed as a percentage of the control. Values are means ± SEM, n = 3. Means not sharing a common letter differ, P < 0.05. (B) CYP24 (left) and CYP27B1 (right) mRNA expression in HMEC cultures treated with ethanol vehicle or 100 nmol/L 25(OH)D3 for 24 h was assessed by real-time PCR and normalized against 18S RNA. Data are expressed as fold of the control cells. Values are means ± SEM, n = 3. *Different from the control, P < 0.01. (C) Mammary cells isolated by collagenase digestion from mice with targeted deletion of CYP27B1 were treated with ethanol vehicle, 100 nmol/L 1,25(OH)2D3 or 100 nmol/L 25(OH)D3 for 48 h prior to determination of CYP24 expression by immunoblotting.

View larger version (20K): [in this window] [in a new window]   FIGURE 5  Expression and regulation of vitamin D hydroxylases in relation to growth in primary cultures derived from human breast. (A) Lysates from PHMEC were immunoblotted with antibodies against VDR or CYP27B1; lysates from PHMEC cultures treated for 24 h with 100 nmol/L 1,25(OH)2D3 or 25(OH)D3 were immunoblotted with antibody against CYP24 or GAPDH (loading control). (B) PHMEC cultures transfected with a CYP24 reporter gene and a normalization gene were treated for 24 h with vehicle, 100 nmol/L 1,25(OH)2D3 or 100 nmol/L 25(OH)D3. RLU for vehicle-treated cells were set to 1 and fold induction by 1,25(OH)2D3 and 25(OH)D3 are shown. Values are means ± SEM, n = 4. Means not sharing a common letter differ, P < 0.05. (C) PHMEC cultures were harvested before and after treatment with vehicle, 100 nmol/L 1,25(OH)2D3 or 100 nmol/L 25(OH)D3 for up to 96 h and cell density was assessed by crystal violet assay. Values are means ± SEM, n = 4, of absorbance at 590 nm, which is proportional to cell density. *Different from the control, P < 0.01.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

These are the first studies to demonstrate that concentrations of 25(OH)D₃ within the range found in the human circulation (35–100nmol/L) are growth inhibitory to nontransformed mammary cells. Further work will be necessary to define how 25(OH)D₃, the majority of which circulates bound to vitamin D binding protein (DBP), gains access to mammary cells. In particular, the relative contributions of free 25(OH)D₃ (which enters cells via diffusion) and DBP-bound 25(OH)D₃ (which enters cells via receptor mediated endocytosis) to the intracellular 25(OH)D₃ pool require clarification. Regardless of the specific mechanism, it is likely that low circulating 25(OH)D₃ subsequent to vitamin D deficiency would reduce substrate availability to CYP27B1 and limit 1,25(OH)₂D₃ production in the mammary gland. A suboptimal supply of 1,25(OH)₂D₃ could result in deregulation of both VDR-mediated gene expression and growth control, a concept supported by data from VDR knockout mice, which exhibit accelerated mammary gland development during puberty and pregnancy (21,26). Furthermore, inhibitory effects of dietary vitamin D and VDR agonists were reported in animal models of breast cancer (27–29). Collectively, these observations provide a potential biological basis for the epidemiologic observations linking vitamin D status in general, and 25(OH)D₃ in particular, to breast cancer risk (17,30).

Particularly relevant to the potential role of vitamin D in breast cancer, both aging and estrogen deficiency are associated with low vitamin D status. Aging reduces vitamin D production in the skin; therefore, elderly individuals are more dependent on dietary and supplemental vitamin D than younger individuals. Estrogen stimulates renal CYP27B1 activity, and estrogen deficiency is associated with low circulating 1,25(OH)₂D₃ (31). Thus, postmenopausal women, the population most at risk for breast cancer, have a high prevalence of marginal vitamin D status (32). Furthermore, it should be noted that the definitions of "adequate," "low," and "deficient" circulating levels of 25(OH)D₃, as well as the intake necessary to sustain appropriate vitamin D status, are currently being reevaluated (1,33).

Our studies also examined the regulation of CYP27B1 and CYP24 gene expression by 1,25(OH)₂D₃ in mammary cells. In all breast-derived cell lines studied, 1,25(OH)₂D₃ induced CYP24, an expected finding because this gene promoter contains a well-characterized VDR responsive region. Consistent with the known negative feedback regulation of 1,25(OH)₂D₃ on its own production, 1,25(OH)₂D₃ downregulated CYP27B1 in nontumorigenic HMEC cultures (Fig. 3C). Surprisingly, 1,25(OH)₂D₃ did not inhibit CYP27B1 gene expression in MCF-7 cells despite the ability of this metabolite to activate VDR and inhibit growth in these cells. This finding suggests that CYP27B1 may be deregulated during transformation, a suggestion that is consistent with data indicating elevated expression of vitamin D–metabolizing enzymes in human breast tumors compared with adjacent normal tissue (20). Follow-up studies on the molecular regulation of CYP27B1 in a defined model of mammary cell transformation are currently in progress to test this hypothesis.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge Cytochroma for the CYP24 antibody and Dr. Loraine Racusen (Johns Hopkins University) for the HKC-8 cells. We thank Dr. Glendon Zinser for technical advice on transfections and real-time PCR and Dr. Matthew Rowling for assistance with mammary cell isolation.

	FOOTNOTES

 
¹ Supported by National Institutes of Health CA103018 and CA96700 to J.W.

² Present address: School of Medicine, University of Pennsylvania, Philadelphia, PA.

⁴ Abbreviations used: [1,25(OH)₂D₃], 1,25-dihydroxycholecalciferol; CYP 450, cytochrome P; HMEC, human mammary epithelial cells [24,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃; [25(OH)D₃], 25-hydroxycholecalciferol; PHMEC, primary HMEC; RLU, relative luciferase units; VDR, vitamin D receptor.

Manuscript received 30 September 2005. Initial review completed 19 October 2005. Revision accepted 13 January 2006.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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				M. M. Turunen, T. W. Dunlop, C. Carlberg, and S. Vaisanen Selective use of multiple vitamin D response elements underlies the 1 {alpha} ,25-dihydroxyvitamin D3-mediated negative regulation of the human CYP27B1 gene Nucleic Acids Res., April 10, 2007; (2007) gkm179v1. [Abstract] [Full Text] [PDF]

				S. S. Tworoger, I-M. Lee, J. E. Buring, B. Rosner, B. W. Hollis, and S. E. Hankinson Plasma 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D and Risk of Incident Ovarian Cancer Cancer Epidemiol. Biomarkers Prev., April 1, 2007; 16(4): 783 - 788. [Abstract] [Full Text] [PDF]

				M. J. Rowling, C. M. Kemmis, D. A. Taffany, and J. Welsh Megalin-Mediated Endocytosis of Vitamin D Binding Protein Correlates with 25-Hydroxycholecalciferol Actions in Human Mammary Cells J. Nutr., November 1, 2006; 136(11): 2754 - 2759. [Abstract] [Full Text] [PDF]

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