Regulation of the CAMP gene by 1,25(OH)2D3 in various tissues

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Abstract

The induction of antimicrobial peptides such as the human cathelicidin, CAMP/hCAP18, by 1,25(OH)2D3 provides a very exciting therapeutic approach in boosting immunity against infectious diseases. To explore the range of cell types and expand the number of cell models for studying the regulation of CAMP gene expression by 1,25(OH)2D3, we treated cell lines from various tissue types and determined CAMP gene expression. Also, we tested additional compounds together with 1,25(OH)2D3 to look for possible cooperative activation of the gene. We identified 1,25(OH)2D3-mediated induction of the CAMP gene in B-cell lymphomas, prostate and endometrial cancer lines and found cooperative activation with the histone deacetylase inhibitor sodium butyrate. The data suggest that regulation of CAMP by 1,25(OH)2D3 is potentially important in a wide range of tissues.

Introduction

Arguably the most potent inducing agent of cathelicidin antimicrobial peptide (CAMP) gene expression is 1,25(OH)2D3 and its analogs [1], [2], [3]. The precursor to 1,25(OH)2D3 is found in the skin and is sequentially metabolized by the liver and kidney to the biologically active form 1,25(OH)2D3. It is a seco-steroid hormone that regulates calcium homeostasis within the body [4]. The genomic actions of 1,25(OH)2D3 are modulated through the vitamin D receptor (VDR) [5]. VDR belongs to a superfamily of nuclear receptors that transmit hormonal signals from the environment and transcriptionally activate genes in response to these signals. Target genes contain hormone response elements (VDREs) in their promoters that heterodimers of VDR and retinoid X receptors (RXR) can bind and activate expression of the target genes [6]. The VDRE of the CAMP gene is located within a short interspersed element (SINE) of the AluSx family that is absent in non-primate mammals [2].

The VDR is expressed in at least 30 different target tissues including bone, kidney, blood, breast, prostate, gut, activated B- and T-lymphocytes, monocytes and keratinocytes [7], [8], [9], [10], [11]. CAMP expression has been described in many of the same tissues [12], [13], [14]. Most dividing cell types, normal and malignant, can express VDR and respond to 1,25(OH)2D3 and would suggest that CAMP expression may potentially be regulated by the vitamin D pathway in many different tissues.

VDR agonists modulate T-cell responses including proliferation, cytokine production, and result in decreased Th1 development and enhanced frequency of Th2 and regulatory T-cells [15], [16]. In addition, VDR agonists inhibit DC differentiation and maturation and promote their apoptosis. Also, 1,25(OH)2D3 reduces the expression of IL-12 and costimulatory molecules increases IL-10 levels. The combined outcome of these effects inhibits DC-dependent T-cell activation [11], [17], [18], [19]. The ability of VDR agonists to promote tolerance in DCs and T-cells has prompted investigators to explore possible therapeutic treatments for a number of human autoimmune diseases [10], [18], [20], [21].

In addition to regulating their response to VDR agonists, macrophages, DCs and T-cells can regulate the production and degradation of 1,25(OH)2D3 suggesting an important biological role for 1,25(OH)2D3 in regulating innate and adaptive immunity [22], [23], [24], [25], [26], [27]. Although 1,25(OH)2D3 and its analogs are important regulators of calcium and bone metabolism, their non-calciotropic activities include inhibition of cell proliferation, promotion of cell differentiation and modulation of immune cell function. These effects have spurred interest in therapeutic applications in a wide variety of diseases [19]. The induction of AMPs by vitamin D3 provides the very exciting possibility of boosting immunity against infectious diseases.

To date, induction of CAMP by 1,25(OH)2D3 has been described in cell lines of various epithelial tissues including squamous cell carcinoma of the tongue (SCC25), lung cancer (Calu-3), colon cancer (HT-29) and keratinocytes (HaCat). Induction was observed in hematopoetic cell lines including myeloid leukemias (U937, HL60, NB4) and in primary tissues and cells including adult and neonatal keratinocytes, skin of normal volunteers, leukocytes (monocytes, neutrophils, macrophages) and bone marrow cells of both normal and leukemic individuals [1], [2], [3].

To explore the range of cell types that show induction of CAMP in response to 1,25(OH)2D3 treatment and expand the number of cell models for studying the regulation of CAMP gene expression by the vitamin D system, we treated cell lines from various tissue types with 1,25(OH)2D3 and examined CAMP gene expression. In addition, we tested a number of additional compounds together with 1,25(OH)2D3 to look for possible cooperative activation of the gene.

Section snippets

Cell culture and reagents

The cell lines examined in this study are summarized in Table 1 and are indicated by bold text. All cell lines were cultured in RPMI 1640 supplemented with 10% FCS, l-glutamine and penicillin/streptomycin (Invitrogen, Carlsbad, CA). For most of the experiments, cells were treated for 24 h with 1,25(OH)2D3 at the concentrations indicated in the figure legends. 1,25(OH)2D3 was synthesized and generously provided by Dr. Milan Uskokovic at Hoffmann-LaRoche Inc. (Nutley, NJ, USA). LPS (1 μg/ml), TPA

Induction of CAMP expression by 1,25(OH)2D3 varies within cell types

Cell lines representing the prostate, lung, breast and colon as well as the monocyte/macrophage and B-cell lineages were tested for expression of CAMP with and without exposure to 1,25(OH)2D3. In the cell lines KCL22, Jurkat, HSB2, H460, H520, HeLa, and SAOS2, expression of CAMP was not detected in either treated or untreated cells (Table 1). On the other hand, CAMP expression was induced in three myeloid leukemias (Fig. 1A and D), three B-cell lymphomas (Fig. 1B), one endometrial (Fig. 1C),

Discussion

In this study, we examined the potential of 1,25(OH)2D3 to induce CAMP gene expression in additional epithelial and hematopoeitic cell lines. These results are summarized along with data from previous studies in Table 1. Not all cell lines from the same tissue showed induction (Table 1). For example, the lung cancer cell lines A549 and Calu-3 showed inducible expression, but H460 and H520 did not. Those cell lines that did not show inducible expression usually had undetectable levels of CAMP

Acknowledgements

We thank Katia Chumakova for technical assistance. We thank Milan Uskokovic for providing reagents. This work was supported by NIH grant CA26038-20, a grant from Leo Pharmaceutical, the Cindy and Allan Horn Foundation, the King Harbor Yacht Club Tom Collier Memorial Fund and the Inger Fund. HPK holds the Mark Goodson Endowed Chair for Cancer Research and is a member of the Jonsson Cancer Center.

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