Direct Regulation of HOXA10 by 1,25-(OH)2D3 in Human Myelomonocytic Cells and Human Endometrial Stromal Cells

Hongling Du, Gaurang S. Daftary, Sasmira I. Lalwani and Hugh S. Taylor

Yale University School of Medicine, New Haven, Connecticut 06520-8063

Address all correspondence and requests for reprints to: Hugh S. Taylor, Division of Reproductive Endocrinology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8063. E-mail: hugh.taylor{at}yale.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vitamin D receptor (VDR) and the functionally active form of its ligand, 1,25-(OH)2D3, have been implicated in female reproduction function and myeloid leukemic cell differentiation. HOXA10 is necessary for embryo implantation and fertility, as well as hematopoeitic development. In this study, we identified a direct role of vitamin D in the regulation of HOXA10 in primary human endometrial stromal cells, the human endometrial stromal cell line (HESC), and in the human myelomonocytic cell line, U937. Treatment of primary endometrial stromal cells, or the cell lines HESC and U937 with 1,25-(OH)2D3 increased HOXA10 mRNA and protein expression. VDR mRNA and protein were detected in primary uterine stromal cells as well as HESC and U937 cells. We cloned the HOXA10 upstream regulatory sequence and two putative vitamin D response elements (VDRE) into luciferase reporter constructs and transfected primary stromal cells and HESC. One putative VDRE (P1: –385 to –434 bp upstream of HOXA10) drove reporter gene expression in response to treatment with 1,25-(OH)2D3. In EMSA, VDR demonstrated binding to the HOXA10 VDRE in the presence of 1,25-(OH)2D3. 1,25-(OH)2D3 up-regulates HOXA10 expression by binding VDR and interacting with a VDRE in the HOXA10 regulatory region. Direct regulation of HOXA10 by vitamin D has implications for fertility and myeloid differentiation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE FUNCTIONALLY ACTIVE form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], acts through its cognate nuclear receptor, vitamin D receptor (VDR). In addition to its well-characterized role in the regulation of calcium metabolism, also exhibits antiproliferative effects and induces differentiation in various cancer cells (1, 2, 3, 4). 1,25-(OH)2D3 is a potent inducer of myeloid-leukemic-cell differentiation. Specifically, 1,25-(OH)2D3 induces murine myeloid leukemia M1 cells to differentiate into cells similar to macrophages (5, 6). Similarly, differentiation of the human myelomonocytic cell line U937 into monocytes/macrophages is also regulated by 1,25-(OH)2D3 (7). Monocytic maturation may be induced by 1,25-(OH)2D3 in blasts from patients with acute myeloid leukemia or myelodysplasia syndrome (8, 9).

Vitamin D also has a role in reproductive function. Treatment of the uterus with 1,25-(OH)2D3 induces decidualization. Female rats fed a vitamin D-deficient diet are capable of reproduction, but overall fertility is reduced by 75%, and litter size is diminished by 30% (10). The effect of vitamin D deficiency is not reversed by calcium supplementation in mice. VDR knockout female mice are unable to reproduce; females demonstrate defects in uterine development and decidualization likely resulting in impaired fertility (11). The synthesis of 1,25-(OH)2D3 from its precursor 25-hydroxyvitamin D[25(OH)D] is catalyzed by 25-hydroxyvitamin D-1{alpha}-hydroxylase [1{alpha}(OH)ase]. 1{alpha}(OH)ase-null mutant mice have uterine hypoplasia and decreased ovarian size; these mice do not ovulate and are infertile due to the combined ovarian and uterine defects (12). Taken together, these data indicate that vitamin D is necessary for myeloid differentiation and fertility.

Hox genes were first recognized as an evolutionarily conserved family of transcription factors critical to the control of early embryonic development. In mammals, the homeobox containing HOX gene family is essential for normal hematopoietic development. Aberrant expression, in particular of HOXA10, is linked to leukemogenesis (13, 14). HOXA10 is strongly expressed in CD34+ human marrow cells and peripheral blood, as well as blood cells from patients with a variety of acute and chronic leukemias. It is markedly down-regulated in CD34-marrow cells, and not expressed in mature neutrophils, monocytes, and lymphocytes (15, 16). The bone marrow of mice reconstituted with HOXA10-transduced bone marrow cells contains a high frequency of a unique progenitor cell with megakaryocytic colony-forming ability and is virtually devoid of either unilineage macrophages or pre-B-lymphoid progenitor cells derived from the transduced cells (17). Expression of HOXA10 in U937 cells also induces differentiation into monocytes, an effect that is similar to that induced by vitamin D treatment. Both HOXA10 and vitamin D induce similar effects in marrow-derived cells.

HOXA10 is expressed in both the embryonic and the adult reproductive tracts, predominantly in the uterus (18). Estrogen and progesterone regulate HOXA10 expression in both the embryonic and the adult reproductive tracts (19, 20). The regulation by sex steroids is direct, as a result of either estrogen receptor or progesterone receptor binding to their respective regulatory elements within the HOXA10 5' regulatory region. HOXA10 expression is up-regulated in response to sex steroids at the time of implantation in the human endometrium (19, 21, 22). HOXA10 is necessary for normal decidualization; Hoxa10 (–/–) mice show defective decidualization (23). Similarly, vitamin D is required for decidualization.

Vitamin D and HOXA10 have a similar effects on the phenotype of marrow-derived cells as well as endometrial cells. Furthermore, HOXA10 can be induced by 1,25-(OH)2D3 in human myeloid leukemic cells (24). We hypothesized that vitamin D and HOXA10 were operative in a common pathway, leading to uterine endometrial receptivity and to monocyte differentiation. Here we determined that 1,25-(OH)2D3 regulated HOXA10 gene expression in human U937 and endometrial cells. We also investigated the molecular mechanism of 1,25-(OH)2D3 activation of the HOXA10 gene promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of HOXA10 by 1,25-(OH)2D3 in the Human Myelomonocytic Cell Line U937
To determine the ability of 1,25-(OH)2D3 to regulate HOXA10 expression in myeloid differentiation, we measured HOXA10 mRNA and protein levels after treatment with 1,25-(OH)2D3 (Fig. 1Go). U937 cells were treated with 1 x 10–7 M 1,25-(OH)2D3. As determined by semiquantitative RT-PCR, at 4 h HOXA10 mRNA levels were markedly increased in 1,25-(OH)2D3-treated cells, consistent with a previously published report (Fig. 1AGo) (25). Figure 1BGo demonstrates the average HOXA10 expression normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression from the same sample. Results are an average of four experiments performed in duplicate. At 16 h, HOXA10 mRNA expression reached a peak (2.7-fold) and remained elevated at 24 h (2.4-fold). The 16- and 24-h results were confirmed by real-time quantitative RT-PCR (Fig. 1CGo). 1,25-(OH)2D3 treatment resulted in a 3.8-fold and 2.4-fold increase of HOXA10 mRNA at 16 and 24 h, respectively. Western blot revealed that HOXA10 protein expression increased at 24 h after treatment with 1,25-(OH)2D3 (Fig. 1DGo). Quantification of HOXA10 protein expression and normalization to actin was performed and the results shown in Fig. 1EGo. Results are an average of four independent experiments. The results suggest that HOXA10 gene expression was regulated by 1,25-(OH)2D3 in U937 cells.



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Fig. 1. 1,25-(OH)2D3 Regulated HOXA10 Expression in U937 Cells

A, U937 cells were treated with or without 1 x 10–7 M 1,25-(OH)2D3 in charcoal-stripped serum-free RPMI. Semiquantitative RT-PCR was performed on total cellular RNA using primers corresponding to HOXA10 and GAPDH. HOXA10 mRNA increased with 1,25-(OH)2D3 treatment at 4, 16, and 24 h. Representative results are shown from a total of four replicates. (–), Negative control lacking RT. C, Results from the untreated cells at time 0 h. B, Densitometric quantification of semiquantitative RT-PCR. HOXA10 mRNA levels normalized to GAPDH are increased by 4 h, reached maximal expression by 16 h, and remain elevated at 24 h. Results are the mean of four replicates ± SE. *, P < 0.01. C, Real-time quantitative RT-PCR was performed using primers corresponding to HOXA10 and ß-actin. Increased HOXA10 mRNA was confirmed at 16 and 24 h after 1,25-(OH)2D3 treatment (n = 4). *, P < 0.01. D, U937 cells were treated with 1 x 10–7 M 1,25-(OH)2D3 or vehicle control (C). Whole cell protein extract was collected and Western blot performed using polyclonal HOXA10 antibody. Actin was used to control for loading. HOXA10 protein was similarly increased by 1,25-(OH)2D3 treatment. Increased protein expression was noted at 24–48 h. Representative results are shown from a total of four replicates. E, Densitometric quantitation of Western blot results was used to quantify HOXA10 protein expression. HOXA10 protein is induced by 1,25-(OH)2D3 after 24 h in U937 cells (n = 4). *, P < 0.01

 
Regulation of HOXA10 by 1,25-(OH)2D3 in Human Endometrial Stromal Cell Line (HESC)
To determine whether 1,25-(OH)2D3 can increase HOXA10 expression in both primary human endometrial stromal cells or an immortalized HESC, these cells were treated with 1,25-(OH)2D3. We determined the dose response and time course of HOXA10 expression after 1,25-(OH)2D3 treatment in primary human endometrial stromal cells using Northern analysis. HOXA10 mRNA expression was induced within 15 min after 1,25-(OH)2D3 treatment (Fig. 2AGo). Expression remained consistently increased to 24 h. Dose response studies were performed using 1-h treatments. Maximal expression was obtained with 1 x 10–7 M 1,25-(OH)2D3 (Fig. 2BGo). Subsequent experiments were conducted using this concentration of 1,25-(OH)2D3. We also quantified HOXA10 mRNA and measured protein levels after treatment with 1,25-(OH)2D3 in HESC (Fig. 2Go, C and D). As determined by real-time quantitative RT-PCR, at 16 h HOXA10 mRNA expression was 4.3-fold higher than that of vehicle-treated cells, consistent with the result using primary endometrial stromal cells. Western blot also revealed that HOXA10 protein levels increased at 24 and 48 h after treatment with 1,25-(OH)2D3. The results suggest that HOXA10 gene expression is regulated by 1,25-(OH)2D3 in endometrial stromal cells and in HESC.



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Fig. 2. 1,25-(OH)2D3 Regulates HOXA10 Expression in Endometrial Cells

A, Primary human endometrial stromal cells were treated with 1 x 10–7 M 1,25-(OH)2D3 and RNA extracted for Northern analysis at various times as shown. HOXA10 expression was measured by laser densitometry of radiograms and normalized to GAPDH mRNA from the same lane. 1,25-(OH)2D3 induced HOXA10 mRNA within 15 min and RNA levels remained elevated after 4 h (n = 6 at each time point). *, P < 0.01. B, Primary human endometrial stromal cells were treated with varying concentrations of 1,25-(OH)2D3 as shown. Maximal HOXA10 expression was induced with 1 x 10–7 M 1,25-(OH)2D3 (n = 6 at each time point). *, P < 0.01. C, HESC were treated with 1 x 10–7 M 1,25-(OH)2D3 or vehicle control in charcoal-stripped CSF-DMEM. Real-time quantitative RT-PCR was performed using primers corresponding to HOXA10 and ß-actin. HOXA10 mRNA levels were normalized to ß-actin. HOXA10 mRNA was increased by 1,25-(OH)2D3 treatment at 16 h (n = 4). *, P < 0.05. D, HESC were treated with or without 1 x 10–7 M 1,25-(OH)2D3. Whole cell protein extract was collected and Western blot was performed using polyclonal HOXA10 antibody. Actin was used to control for loading. HOXA10 protein was also increased by 1,25-(OH)2D3 treatment in HESC. A significant increase was noted by 24 h.

 
VDR Expression in U937 and Endometrial Cells
To determine whether the classic receptor mediated mechanism (26, 27, 28, 29, 30, 31) was potentially used in the induction of HOXA10 by 1,25-(OH)2D3, we identified the expression of VDR in several cell lines; these included the U937 cell line, the endometrial epithelial adenocarcinoma cell line Ishikawa, primary human endometrial stromal cells, and the HESC endometrial stromal cell line. RT-PCR was used to identify VDR mRNA in primary endometrial stromal cells (Fig. 3AGo). RT-PCR and Western blot analysis showed that VDR was expressed in the cell lines U937 and HESC, but not in the Ishikawa cells. (Fig. 3Go, B and C; RT-PCR and Western, respectively). The breast cancer cell line MCF-7 was used as positive control (32). The results suggest that 1,25-(OH)2D3 may regulate HOXA10 expression through the VDR. The HESC cell line was subsequently used to evaluate the mechanism of vitamin D response.



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Fig. 3. U937 and HESC Express VDR

A, Total RNA was extracted from primary human endometrial stromal cells (PESC), and RT-PCR was performed to detect the expression of VDR. Primary human endometrial stromal cells express VDR. RNA obtained from MCF-7 cells was used as a positive control. Representative results are show from three replicates. GAPDH was coamplified as a control for loading. B, Total RNA was extracted from the Ishikawa, U937, HESC and MCF-7 cell lines; RT-PCR was performed to detect the expression of VDR. VDR was detected in U937 and HESC cell line, but not the Ishikawa cell line. RNA obtained from MCF-7 cells was used as a positive control. Representative results are show from three replicates. C, Whole cell protein was extracted from the Ishikawa, U937, and HESC cell line. Western blot was performed to detect the expression of VDR, and confirmed the results of the RT-PCR. MCF-7 cell protein extract was used as positive control. Representative results are show from three replicates.

 
Induction of HOXA10 Promoter Activity by 1,25-(OH)2D3
To determine whether 1,25-(OH)2D3 directly regulated transcription of HOXA10 gene expression, HESC cells were transfected with an artificial reporter construct containing the HOXA10 promoter and 5' regulatory region. Approximately 1.0 kb of the HOXA10 5' sequence (–15/–985 from the translation start site) was amplified by PCR and cloned into pGL3-basic vector (Promega, Madison, WI) (Fig. 4AGo). After transient transfection for 24 h, cells were treated for 48 h with 1,25-(OH)2D3. The pRL-TK vector was cotransfected into HESC as a control for transfection efficiency and used for normalization. In transactivation assays, 1,25-(OH)2D3 induced a 2.7-fold increase in luciferase activity driven by the HOXA10 wild-type 5' promoter sequence in HESC. Figure 4BGo demonstrates average fold increase in normalized luciferase activity after vitamin D treatment. Results are an average of four experiments and statistically significant (t test; P < 0.05). The results demonstrate the existence of a putative VDRE 5' of the HOXA10 coding sequence.



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Fig. 4. Transcriptional Activation of the HOXA10 Promoter by 1,25-(OH)2D3 in HESC

A, Approximately 1 kb of the human HOXA10 5' regulatory region was amplified by PCR and cloned into the pGL3-basic vector. The schematic illustrates the construct employed. Site-directed mutagenesis was used to introduce mutation into a putative VDRE in the 1-kb construct. The sequence and location of the mutation are shown. B, The HOXA10 5' regulatory sequence reporter construct was transiently transfected into HESC. The cells were treated with 1 x 10–7 M 1,25-(OH)2D3 or vehicle control for 48 h. 1,25-(OH)2D3 treatment induced a 2.7-fold increase in luciferase activity (n = 4). *, P < 0.05. No significant increase in luciferase activity was obtained after 1,25-(OH)2D3 treatment using the mutated regulatory element.

 
A HOXA10 VDRE Drives Transcription in Response to 1,25-(OH)2D3
To determine whether 1,25-(OH)2D3/VDR directly regulates the transcription of HOXA10 gene expression, HESC cells were transfected with artificial heterologous promoter constructs containing HOXA10 upstream regulatory regions. We performed sequence analysis of the HOXA10 gene promoter and the region 5 kb upstream of the transcription start site and identified two putative VDREs, P1 and P2 (Fig. 5AGo) located within the previously described 1 kb 5' regulatory region. These putative VDRE and immediately adjacent sequences were cloned into pGL3-promotor. The well-characterized mouse osteopontin (MOP) VDRE sequence cloned into the pGL3-promoter vector was used as a positive control. After transient transfection for 24 h, cells were treated for 48 h with 1,25-(OH)2D3. The pRL-TK vector was cotransfected into HESC as a control for transfection efficiency. In transactivation assays, the MOP control sequence resulted in a 1.7-fold increase in luciferase expression induced by 1,25-(OH)2D3 in HESC (Fig. 5BGo). A similar 2.4-fold increase was induced by the P1 HOXA10 VDRE (Fig. 5BGo). We did not observe an increase in luciferase activity driven by the P2 sequence in HESC. Primary human stromal cells were also transfected with the pGL3-promoter/P1 and P2 vectors (Fig. 5CGo). Treatment of primary stromal cells with 1,25-(OH)2D3 resulted in a 5-fold increase in luciferase activity driven by the P1 VDRE. The P2 containing vector showed no response to 1,25-(OH)2D3 treatment in these cells. Additionally, Ishikawa cells were used in identical experiments (Fig. 5DGo). Predictably, no increase in luciferase activity was observed using either the P1 or the P2 containing reporter vectors in Ishikawa cells, consistent with the lack of VDR. Empty pGL3-promoter vector luciferase activity was not altered by the presence of 1,25-(OH)2D3 in any cell type (Fig. 5Go, C and D). The results demonstrate the existence of a putative VDRE in the region –385 to –434 bp upstream of the HOXA10 coding sequence.



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Fig. 5. A Putative VDRE Drove Luciferase Activity in Response to 1,25-(OH)2D3

A, A schematic representation of 5' region in HOXA10 gene is shown. The putative VDREs identified in the 5' HOXA10 regulatory region are labeled P1 and P2. The sequence and location relative to the HOXA10 translation start are shown below the schematic. The sequence of the MOP-positive control sequence is also given. The P1 and P2 oligonucleotides were used in both EMSA and in reporter constructs as shown. B, The HOXA10 putative VDREs were cloned into the pGL3-promoter vector and were transiently transfected into HESC. The cells were treated with 1 x 10–7 M 1,25-(OH)2D3 or vehicle control for 48 h. A 2.4-fold and 1.7-fold increase in luciferase activity were obtained when the P1 or MOP VDREs were used, respectively. *, Statistically significant difference from vehicle-treated control at P < 0.001 using paired t test. P2 failed to drive a response to vitamin D treatment. All experiments were repeated at least four times. C, The putative VDRE containing constructs were used to transfect primary stromal cells. P1 drove a 5-fold increase in luciferase activity in response to 1,25-(OH)2D3. In these cells neither empty plasmid (pGL3) nor P2 induced reporter activity in response to treatment. *, Statistically significant difference from vehicle-treated control at P < 0.001 using paired t test. D, Ishikawa endometrial epithelial cells were similarly transfected. None of the constructs responded to treatment, consistent with the lack of VDR in this cell line (n = 4 in each luciferase assay).

 
VDR Binds to the HOXA10 VDRE
To determine whether the HOXA10 VDRE bound the VDR in the presence of 1,25-(OH)2D3, EMSAs were performed as shown in Fig. 6AGo. As positive control, MOP was incubated with HESC (lanes 1 and 2) or MCF-7 cell nuclear extract (lane 7) known to contain active VDR (32). Double-stranded P1 and P2 were incubated with nuclear extract from HESC cells previously treated with or without 1,25-(OH)2D3. A specific complex from HESC nuclear extract was observed with the P1 probe (lanes 3 and 4). MOP or P1 VDRE binding was increased significantly when nuclear extract from vitamin D-treated HESC was used. In contrast, no shifted complex was observed using probe P2 (lanes 5 and 6), consistent with the lack of activity driven by this putative VDRE in the luciferase reporter assay.



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Fig. 6. EMSA Showing the Specific Binding of VDR to the HOXA10 VDRE (P1)

A, HESC cells were treated with or without 1,25-(OH)2D3 for 48 h and nuclear extract collected for use in EMSA. Lanes 1 and 2 demonstrate interaction of the labeled positive control MOP probe with nuclear extract from HESC. The arrow indicates the shifted complex. Without treatment with 1,25-(OH)2D3 minimal binding is noted (lane 1). Lane 2 demonstrates the interaction of 32P-labeled probes MOP with nuclear extract from HESC treatment with 1,25-(OH)2D3; significant binding is observed. Similarly, the P1 probe shows increased binding in the presence of nuclear extract obtained from 1,25-(OH)2D3-treated cells (lanes 3 and 4). Lanes 5 and 6 demonstrate lack of a shifted complex using the 32P-labeled P2 probe with nuclear extract from HESC, irrespective of 1,25-(OH)2D3 treatment. Lane 7 is a positive control demonstrated the interaction of 32P-labeled MOP VDRE with nuclear extract from MCF-7 cells. Lanes 8 and 9 demonstrate results using the mutated probe. P1 probe containing a mutation in the putative VDRE fails to bind nuclear extract. Lane 10 demonstrated further retarded mobility of the shifted complex (supershift) using wild-type P1 probe, VDR monoclonal antibody and 1,25-(OH)2D3-treated HESC nuclear extract. The arrowhead indicates the supershifted complex. The mutated P1 probe demonstrates neither a shifted or supershifted complex under these conditions (lane 11). Representative results are shown from a total of four replicates. B, HESC cells were treated with 1,25-(OH)2D3 or vehicle control for 48 h and nuclear extract collected for use in EMSA. Before performing EMSA, nuclear extract was incubated with 1,25-(OH)2D3 or vehicle control for 1 h on ice. Lane 1 demonstrates absent or minimal of binding using the P1 probe and nuclear extract from HESC without pretreatment with 1,25-(OH)2D3. Treatment of the cell-free nuclear extract with 1,25-(OH)2D3 induced binding of protein to labeled probe (lane 2). Lanes 3 and 4 demonstrate the interaction of P1 with nuclear extract from HESC treatment with 1,25-(OH)2D3. The results demonstrate increased binding after treatment of cells or cell-free nuclear extract with 1,25-(OH)2D3. Representative results are shown from a total of four replicates. Arrow indicates the shifted complex.

 
To further define the specificity of P1/VDR interaction, a mutation was introduced into the putative P1 VDRE that altered the two GGT sequences in each half-site (–417/–415 and –408/–406) to AAC. The mutated probe failed to result in a shifted complex in the presence of nuclear extract (lanes 8 and 9). To confirm that the shifted complex included VDR, anti-VDR antibody was used in EMSA. A supershift (DNA-protein-antibody) complex from HESC nuclear extract was observed using the P1 probe (lane 10). No supershifted complex was observed using the mutated P1 probe (lane 11). Primary stromal cell nuclear extract was also used in EMSA. A shifted complex was identified identical with that observed using HESC (data not shown). The results suggest that the P1 sequence in the HOXA10 gene is a novel VDR binding element, through which 1,25-(OH)2D3 induces HOXA10 expression.

To determine whether 1,25-(OH)2D3 induced VDR binding or alternatively increasing VDR expression in the cells, we performed EMSA using nuclear extract from 1,25-(OH)2D3-treated and untreated cells. As demonstrated in Fig. 6BGo, nuclear extract from untreated cells (lane 1) is unable to shift the labeled probe. Treatment of the cell-free nuclear extract with 1,25-(OH)2D3 resulted in a shifted complex (lane 2), suggesting an effect on VDR binding rather than expression. As previously demonstrated, nuclear extract obtained from 1,25-(OH)2D3 pretreated cells (lane 3) retards the mobility of the P1 VDRE. Similarly, pretreatment of the cells and subsequent treatment of extract results in an equivalent shifted complex (lane 4).

Identification of a Functional VDRE in the HOXA10 Promoter
We determined whether the VDRE sequence, identified above in the in HOXA10 gene, regulates the previously described 1,25-(OH)2D3 responsiveness of the 1-kb region 5' of HOXA10. HESC cells were transfected with the artificial heterologous construct pGL3-basic containing the HOXA10 promoter region containing the mutation that eliminated VDR binding in EMSA. The mutagenesis substituted AAC for GGT (–417/–415 and –408/–406) as shown in Fig. 4Go. After transient transfection for 24 h, cells were treated for 48 h with 1,25-(OH)2D3. The pRL-TK vector was cotransfected into HESC as a control for transfection efficiency. In transactivation assays, 1,25-(OH)2D3 did not induce reporter activity driven by the HOXA10 mutated promoter sequence (P > 0.05) (Fig. 4BGo). The results demonstrate the putative VDRE (P1) in the HOXA10 promoter is responsible for the 1,25-(OH)2D3 responsiveness of the HOXA10 gene.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In addition to sex steroid hormones, the classic regulators of reproduction, vitamin D also modulates reproductive processes in the human female; its nuclear receptor has been identified in the uterus, oviduct, ovary, placenta, and fetal membranes (33). Human uterine endometrial cells and decidual cells synthesize 1,25-(OH)2D3 (34, 35, 36). 1{alpha}-Hydroxylase and VDR expression increase in first- and second-trimester placenta and decidua compared with nonpregnant endometrium (35). Patients with pseudo-vitamin D deficiency rickets have an inherited defect in the gene for 1{alpha}-hydroxylase, are unable to convert 25(OH)D3 to 1,25-(OH)2D3 and defective decidualization (36, 37, 38, 39, 40). 1,25-(OH)2D3, its synthetic enzymes and receptor, are all present at the fetal-maternal interface.

Similarly 1,25-(OH)2D3 regulates differentiation of the rodent uterus. 1,25-(OH)2D3 has a physiological role in endometrial cell differentiation into decidual cells, a crucial step in the process of blastocyst implantation. Intraluminal injection of the female rat uterus at d 5 of pseudopregnancy with 1,25-(OH)2D3 significantly increases uterine weight and induces the decidual reaction (36, 41, 42). Vitamin D deficiency reduces mating success and fertility in female rats (10, 43). Both VDR and 1{alpha}-hydroxylase knockout female mice are infertile (12, 45, 46). VDR knockout mice show inadequate uterine development and infertility. 1{alpha}-Hydroxylase knockout mice are anovulatory and infertile. Taken together, these data show that vitamin D has an essential role in fertility, necessary for differentiation of decidual cells.

1,25-(OH)2D3 also potently inhibits cellular proliferation as well as induces differentiation of myeloid leukemic cells; vitamin D treatment leads to differentiation of these cells to macrophages. Specifically the human myelomonocytic cell line U937 responds to 1,25-(OH)2D3 treatment (25). A number of genes are up-regulated by 1,25-(OH)2D3 during myeloid differentiation including HOXA10. Similarly, HOXA10 is important for human hematopoietic development; aberrant expression contributes to impaired differentiation and increased proliferation of human hematopoietic progenitor cells (13). HOXA10 overexpression is also associated with cell growth and several leukemias and also has an antidifferentiative effect in early progenitors. HOXA10 expression is observed in chronic myelogenous leukemia, however, appears to be reduced in accelerated phase and blast crisis, particularly lymphoid blast crisis (15). Ectopic expression of HOXA10 by transient transfection is sufficient to drive a portion of U937 cells to terminally differentiate into the monocyte phenotype (25, 47). Aside from the well-known role of vitamin D in calcium homeostasis, its regulation of HOX gene expression may be a common pathway by which this hormone induces differentiation.

The mechanism of HOXA10 action in leukemic cell differentiation is, at least in part, mediated through p21 regulation. Genes up-regulated by 1,25-(OH)2D3 during myeloid differentiation include HOXA10 and the cyclin-dependent kinase inhibitor p21. Differentiation of U937 cells to monocyte/macrophages after 1,25-(OH)2D3 treatment is caused in part by increased levels of cyclin-dependent kinase inhibitors (7, 48). In U937 cells, p21 expression leads to G1 arrest and differentiation; similarly increased expression of HOXA10 induced by 1,25-(OH)2D3 leads to G1 arrest and subsequent differentiation to the monocytic cell type. HOXA10 protein directly regulates p21 in a complex with PBX1 and MEIS1, binding the p21 regulatory region and activating transcription (7). HOXA10 also interacts with negative cis elements to repress gene transcription together with PBX1 and histone deacetylase 2 (50, 51). These results suggested that HOXA10 exerts its various cellular effects through p21 as well as other target genes. Collectively, these data show that U937 cell differentiation induced by 1,25-(OH)2D3 may be mediated through direct regulation of HOXA10 gene expression and subsequent p21 expression.

We have identified a novel VDRE. The spacing of the half-sites in steroid superfamily response elements has an essential role in specifying the receptor dimer that binds. The sequence of the steroid hormone half elements are also determinants the relative affinity for the receptor (52, 53, 54, 55). Additionally, specific nucleotides in the VDRE influence the conformation of bound VDR/RXR heterodimers and determine functional activity. Minor changes in the nucleotide sequence of half-elements can change a negative VDRE that suppresses transcription into a positive VDRE that activates in a 1,25-(OH)2D3-dependent manner (56, 57). Osteocalcin and osteopontin are two well-characterized 1,25-(OH)2D3-regulated genes in osteoblasts. Small nucleotide differences between osteopontin and osteocalcin VDREs affect DNA binding and transactivation in response to 1,25-(OH)2D3 (58). The HOXA10 VDRE and the osteopontin VDRE share 60% nucleotide sequence conservation (Table 1Go). These significant differences may reflect a distinct regulatory role of the HOXA10 VDRE in tissues where vitamin D is not primarily involved the regulation of calcium metabolism.


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Table 1. Comparison of VDRE Sequences

 
HOX genes impart segmental identity to developmental axes, including the developing reproductive tract (59, 60, 61, 62). HOXA10 expression is important for development of the uterus and essential for endometrial development, allowing uterine receptivity to implantation (18, 20, 63). Previous studies from our laboratory have shown that regulated maternal HOXA10 expression is necessary for fertility (19, 64). Aberrant HOXA10 expression in patients with infertility confirms its function in human implantation (65, 66, 67). Altered vitamin D signaling may impact HOXA10 expression and human fertility.

HOXA10 is regulated by estrogens and progestins (19, 20). Sex steroids, vitamin D, and HOXA10 are each necessary for normal decidualization. An estrogen response element in the human HOXA10 gene was identified, which mediates differential ligand-specific estrogen-responsive transcriptional activation (22). In this study, we showed that HOXA10 expression was also up-regulated by 1,25-(OH)2D3 in human endometrial stromal cells. Cross talk between sex steroids and vitamin D may converge in regulation of HOXA10 through its estrogen response element and VDRE, respectively.

In summary, 1,25-(OH)2D3 induces HOXA10 transcription through VDR binding to a VDRE in the HOXA10 gene 5' region. Vitamin D regulation of fertility and human myelomonocytic differentiation likely involves the direct transcriptional activation of HOXA10. Vitamin D may induce differentiation of diverse tissues through activation of classic developmental modulators such as HOX genes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
The human myelomonocytic cell line U937, HESC cells (68), and human endometrial adenocarcinoma cell line Ishikawa, were generous gifts of Nancy Berliner, Charles J. Lockwood, and Richard Hochberg (all at Yale University), respectively. U937 cells were cultured in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA), supplemented with 10% charcoal-stripped calf serum and 1% penicillin/streptomycin and 1% sodium pyruvate. HESC were maintained in a phenol-red-free DMEM, Ham’s F-12 (Sigma, St. Louis, MO), supplemented with 10% charcoal-stripped calf serum, 1% penicillin/ streptomycin, and 1% sodium pyruvate. Ishikawa were maintained in MEM (Sigma), supplemented with 10% charcoal-stripped calf serum, 1% penicillin/ streptomycin and 1% sodium pyruvate.

RT-PCR
Total RNA was isolated from U937 and HESC using RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. A total of 0.5 µg oligo(deoxythymidine) (Invitrogen) and 25 U reverse transcriptase avian myeloblastosis virus (AMV) (Roche Molecular Biochemicals, Indianapolis, IN) were used to synthesize the first-strand cDNA from 2 µg total RNA in a reaction volume of 20 µl. PCR amplification was performed using 2 µl of the cDNA product and primers for HOXA10, VDR, and GAPDH as listed in Table 2Go. Semiquantitative RT-PCR for HOXA10 mRNA expression followed our previously described protocol (44). Two micrograms of total RNA were reverse-transcribed in a 20-µl reaction mixture containing 0.5 mM each of deoxy (d) ATP, dCTP, dGTP, and dTTP; 20 pmol oligo(deoxythymidine); 20 U of RNasin inhibitor, 10 U of AMV-reverse transcriptase, and 1x AMV-reverse transcriptase buffer (42 C for 60 min; 95 C for 5 min). The PCR was conducted using 2 µl of cDNA in a total volume of 25 µl containing 1x PCR buffer, 1 µM each of primers (95 C for 30 sec; 61 C for 30 sec; 72 C for 45 sec, 25 cycles). PCR products and molecular weight markers were separated on 1.5% agarose gels and visualized by ultraviolet light. Densitometric quantitation of RT-PCR was analyzed by Kodak 1D scientific imaging system software (Eastman Kodak, Rochester, NY). The intensity of each band was normalized to the corresponding GAPDH band to compare values in a semiquantitative manner.


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Table 2. RT-PCR Primers

 
Quantitative Real-Time PCR
Quantitative real-time RT-PCR was performed using the Lightcyle SYBR Green RT-PCR kit (Roche). RNA was reverse-transcribed for 30 min at 61 C. PCR for HOXA10 was performed for 45 cycles at 95 C for 2 sec; 65 C for 5 sec; 72 C for 18 sec. PCR for control ß-actin was performed for 45 cycles of 95 C for 2 sec; 61 C for 5 sec; 72 C for 18 sec. Primers are listed in Table 2Go. The Roche Light Cycler monitored the increasing fluorescence of PCR products during amplification. A quantitative standard curve was then created. Expression was quantified using the Roche Light Cycler and adjusted to the quantitative expression of ß-actin from the same sample. Melting curve analysis was conducted to determine the specificity of the amplified products and to ensure the absence of primer-dimer formation. All products obtained yielded the predicted melting temperature.

Northern Analysis
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Twenty micrograms of RNA were loaded per lane in a 1% agarose/0.66 M formaldehyde gel and, after partial hydrolysis with NaOH, transferred onto nylon membranes and immobilized using UV irradiation. Northern blot hybridizations were performed as described previously using in vitro transcription with [{alpha}-32P]-deoxyuridine triphosphate (21, 49). A 103-bp fragment of the 3' untranslated region of human HOXA10 was used as the template for generating the riboprobes. [32P]-labeled probe/mRNA hybrids were visualized by autoradiography. Equal loading of samples was verified by stripping the membrane and reprobing with a probe to GAPDH. The experiment was performed in triplicate and repeated. Quantification was performed using laser densitometry (Molecular Dynamics, Sunnyvale, CA)

Western Blot
Nuclear protein was extracted from U937 and HESC using Nuclear Extract Kit (Activemotif) according to manufacturer’s protocol. Equal amounts of protein (60 µg for HOXA10 and 30 µg for VDR) were electrophoresed through 4–15% polyacrylamide gels (Bio-Rad, Hercules, CA) at 160 V for 50 min and transferred onto Immun-Blot polyvindylidene difluoride membranes (Bio-Rad) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 100 V for 1 h. After incubation in blocking buffer (1x PBS, 0.2% Tween 20, 5% milk), the blot was incubated individually with goat polyclonal HOXA10 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) dilution 1:200, overnight at 4 C and goat polyclonal actin antibody (Santa Cruz) dilution 1:1000, room temperature for 1 h, or incubated with rabbit VDR polyclonal antibody (Santa Cruz) dilution 1:500 overnight at 4 C. After washing, the membranes were incubated for 1 h with peroxidase-label antigoat IgG or antirabbit IgM secondary antibody (Vector Laboratories, Burlingame, CA) diluted (5 µg/ml) in the blocking buffer. After a second series of washing, bound antibody complexes were visualized using the Chemiluminescent Reagent Plus Kit (PerkinElmer Life Sciences, Foster City, CA) and subsequent exposure to X-OMAT film (Kodak). Specific bands on the autoradiograms were quantitated by densitometry.

Construction of Plasmid for Promoter Analysis and in Vitro Mutagenesis
Approximately 1.0 kb of the HOXA10 promoter sequence (–15/–985) was amplified by PCR and cloned into pGL3-basic vector (Promega). The fragment was generated by PCR from human genomic DNA (Promega) using HOXA10 5' regulatory region-specific primers with added restriction sites. The primers were designed using the GenBank database (NT_079592). The sequences of the primers were as follows: 5'-GCCTCGAGTCCCCGAAATGACTGTGG-3'; 5'-CGGGGTACCAGCCCTTTCTGGCTGACA-3'. PCR was performed as follows: 95 C for 60 sec; 60 C for 60 sec; 72 C for 90 sec, 35 cycles. The QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used for in vitro mutagenesis. The HOXA10 promoter was mutated using a 48-oligomer oligonucleotide (location as indicated in Fig. 4Go) 5'-CCGCGCAGTTAACAAGTGGAACGTTTATAACGCGCGCCCAGTCTGCC-3'. The mutagenesis reaction was carried out according to the manufacturer’s protocol and the mutation was confirmed by sequencing.

Transfection and Luciferase Assays
HESC, grown to 60–70% confluence in 24-well plates, were transfected using TransIT-LT1 (Mirus, Madison, WI) with 0.4 µg of either pGL3-basic-HOXA10 Promoter, pGL3-basic-HOXA10 mutated Promoter, pGL3-MOP, pGL3-P1 or pGL3-P2, containing putative VDRE sequences. The construct design is shown in Fig. 4AGo. All cells were cotransfected with 10 ng pRL-TK to control for transfection efficiency. HESCs were transfected with empty pGL3-basic vector and pGL3-promoter vector as controls. After 4 h, the media were changed and cells were incubated for an additional 20 h. Then cells were treated with or without 1 x 10–7 M 1,25-(OH)2D3 for 48 h. The cells were rinsed with cold PBS and lysed with 1x Reporter Lysis Buffer (Promega). The lysate was collected after two freeze/thaw cycles. Luciferase activity was measured using the luciferase reagent kit (Promega). Transfections were performed in duplicate and experiments were repeated five times. The MOP VDRE was used as a positive control (58).

EMSA
Complementary oligonucleotides corresponding to the sequences shown in Fig. 5AGo were annealed and end labeled with 32P-dATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (New England BioLabs, Ipswich, MA) and purified with MicroSpin G-25 columns (Amersham Pharmacia Biotech, Piscataway, NJ). The site of mutation in the P1 probe is located at –417/–415 and –408/–406 as shown in Fig. 4Go. Nuclear extract was obtained from HESC using Nuclear Extract Kit (Activemotif, Carlsbad, CA) according to the manufacturer’s protocol. MCF-7 nuclear extract was purchased from Activemotif. Binding reactions were performed as previously described (70). In brief, 25 µl of mixture of 10 µg nuclear extract and 80,000 cpm 32P-labeled oligonucleotides were incubated for 40 min at 37 C. The resultant protein-DNA complexes were separated on a 5% polyacrylamide gel (acrylamide/bisacrylamide, 29:1 for 3 h at 180 V in 0.5x TBE buffer (1x TBE is 50 mM Tris, 50 mM boric acid, and 1 mM EDTA) at 4 C. To confirm the identity of VDR in the shifted complex, 10 µg nuclear extract protein were incubated with 5 µg antivitamin D receptor monoclonal antibody (Affinity BioReagents) at 4 C for 1 h, followed by a 40-min incubation with labeled oligonucleotides at 37 C. Experiments were performed using nuclear extract incubated with either 1x 10–7 M 1,25-(OH)2D3 or vehicle control for 1 h on ice before adding probe, as described. The gel was dried under vacuum at 80 C, exposed overnight on X-OMAT film, and subsequently developed.

Statistical Analysis
One-way repeated measures ANOVA was performed on data obtained from the RT-PCR and Western blot. The paired t test was performed to compare promoter activity.


    FOOTNOTES
 
This work was supported by supported by National Institutes of Health Grants HD36887 and ES10610.

First Published Online May 19, 2005

Abbreviations: AMV, Avian myeloblastosis virus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HESC, the human endometrial stromal cell line; MOP, mouse osteopontin; VDR, vitamin D receptor; VDRE, vitamin D response elements.

Received for publication August 26, 2004. Accepted for publication May 10, 2005.


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