Peter J. Malloy

Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor.

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

The vitamin D receptor gene start codon polymorphism : A functional analysis of FokI variants

The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where “f” indicates the presence of the restriction site and the first ATG, while “F” indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F‐VDR) being three amino acids shorter than the f variant (f‐VDR). To determine whether there are functional differences between the f‐VDR and the F‐VDR, we studied the two VDR forms expressed in COS‐7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f‐VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]‐1,25‐dihydroxyvitamin D3 ([3H]‐1,25(OH)2D3) (Kd = 131 ± 78 pM, f‐VDR; Kd = 237 ± 190 pM, F‐VDR; p = 0.24); however, a 2‐fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24‐hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose‐response (0.1–10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF,Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24‐hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f‐VDR and F‐VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.

Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness.

Mutations in the vitamin D receptor (VDR) result in target organ resistance to 1alpha,25-dihydroxyvitamin D [1,25(OH)2D3], the active form of vitamin D, and cause hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR). We analyzed the VDR of a patient who exhibited three genetic diseases: HVDRR, congenital total lipodystrophy, and persistent mullerian duct syndrome. The patient was treated with extremely high dose calcitriol (12.5 microg/d) which normalized serum calcium and improved his rickets. Analysis of [3H]1,25(OH)2D3 binding in the patients cultured fibroblasts showed normal abundance of VDR with only a slight decrease in binding affinity compared to normal fibroblasts when measured at 0 degrees C. The patients fibroblasts demonstrated 1,25(OH)2D3-induction of 24-hydroxylase mRNA, but the effective dose was approximately fivefold higher than in control cells. Sequence analysis of the patients VDR gene uncovered a single point mutation, H305Q. The recreated mutant VDR was transfected into COS-7 cells where it was 5 to 10-fold less responsive to 1,25(OH)2D3 in gene transactivation. The mutant VDR had an eightfold lower affinity for [3H]1,25(OH)2D3 than the normal VDR when measured at 24 degrees C. RFLP demonstrated that the patient was homozygous for the mutation while the parents were heterozygous. In conclusion, we describe a new ligand binding domain mutation in the VDR that causes HVDRR due to decreased affinity for 1,25(OH)2D3 which can be effectively treated with extremely high doses of hormone.

Genetic Disorders and Defects in Vitamin D Action

Two rare genetic diseases can cause rickets in children. The critical enzyme to synthesize calcitriol from 25-hydroxyvitamin D, the circulating hormone precursor, is 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). When this enzyme is defective and calcitriol can no longer be synthesized, the disease 1alpha-hydroxylase deficiency develops. The disease is also known as vitamin D-dependent rickets type 1 or pseudovitamin D deficiency rickets. When the VDR is defective, the disease hereditary vitamin D-resistant rickets, also known as vitamin D-dependent rickets type 2, develops. Both diseases are rare autosomal recessive disorders characterized by hypocalcemia, secondary hyperparathyroidism, and early onset severe rickets. In this article, these 2 genetic childhood diseases, which present similarly with hypocalcemia and rickets in infancy, are discussed and compared.

Hereditary 1,25-dihydroxyvitamin D resistant rickets.

Publisher Summary Vitamin D, the primary regulator of calcium homeostasis in the body, is particularly important in skeletal development and in bone mineralization. Hereditary 1,25-dihydroxyvitamin-D-resistant rickets (HVDRR) is a rare genetic disease that is due to a generalized resistance to 1,25(OH)2D3. HVDRR is caused by heterogeneous mutations in the vitamin D receptor (VDR) gene that cause loss of function of the receptor ultimately leading to complete or partial target organ resistance to 1,25(OH)2D3. HVDRR is manifested by a constellation of signs and symptoms caused by a generalized resistance to 1,25(OH)2D and by a loss of ligand-dependent and ligand-independent actions of the VDR. The main features of HVDRR are severe rickets with osteomalacia, hypocalcemia, secondary hyperparathyroidism, hypophosphatemia, and elevated alkaline phosphatase. The major manifestation of the defective VDR on the vitamin D endocrine system is to decrease intestinal calcium and phosphate absorption that results in decreased bone mineralization and rickets. The VDR is also expressed in a wide variety of tissues, including kidney, skin, liver, pancreas, muscle, breast, prostate, adrenal, thyroid, and cells of mesenchymal or hematopoietic origin. The biochemical and genetic analysis of the VDR in HVDRR patients has yielded important insights into the structure and function of the receptor in mediating 1,25(OH)2D3 action. A concerted investigative approach of HVDRR at the clinical, cellular, and molecular level has proven exceedingly valuable in understanding the mechanism of action of 1,25(OH)2D3 and improving the diagnostic and clinical management of this rare genetic disease.

Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer

Calcitriol, the hormonally active form of Vitamin D, inhibits the growth and development of many cancers through multiple mechanisms. Our recent research supports the contributory role of several new and diverse pathways that add to the mechanisms already established as playing a role in the actions of calcitriol to inhibit the development and progression of prostate cancer (PCa). Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in the inhibition of PCa cell growth by increasing the expression of the cell cycle inhibitor p21. Calcitriol inhibits the prostaglandin (PG) pathway by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, the inhibition of the PG pathway may add to the ability of calcitriol to prevent and inhibit PCa development and growth. The combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of a family of phosphatases that are negative regulators of MAP kinases. Calcitriol induces MKP5 expression in prostate cells leading to the selective dephosphorylation and inactivation of the stress-activated kinase p38. Since p38 activation is pro-carcinogenic and is a mediator of inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, contributes to the chemopreventive activity of calcitriol in PCa. Mullerian Inhibiting Substance (MIS) has been evaluated for its inhibitory effects in cancers of the reproductive tissues and is in development as an anti-cancer drug. Calcitriol induces MIS expression in prostate cells revealing yet another mechanism contributing to the anti-cancer activity of calcitriol in PCa. Thus, we conclude that calcitriol regulates myriad pathways that contribute to the potential chemopreventive and therapeutic utility of calcitriol in PCa.

Interaction of the Vitamin D Receptor with a Vitamin D Response Element in the Müllerian-Inhibiting Substance (MIS) Promoter: Regulation of MIS Expression by Calcitriol in Prostate Cancer Cells

Calcitriol (1,25-dihydroxyvitamin D(3)) inhibits the growth of a variety of cancer cells including human prostate cancer. Müllerian-inhibiting substance (MIS) also exhibits antiproliferative and proapoptotic actions on multiple cancer cells including human prostate cancer. In this study, we investigated whether calcitriol regulated MIS expression in prostate cancer, an action that might contribute to its antiproliferative activity. We identified a 15-bp sequence, GGGTGAgcaGGGACA, in the MIS promoter that was highly similar to direct repeat 3-type vitamin D response elements (VDREs). The human MIS promoter containing the putative VDRE was cloned into a luciferase reporter vector. In HeLa cells transfected with the vitamin D receptor (VDR), MIS promoter activity was stimulated by calcitriol. Coexpression of steroidogenic factor 1, a key regulator of MIS, increased basal MIS promoter activity that was further stimulated by calcitriol. Mutation or deletion of the VDRE reduced calcitriol-induced transactivation. In addition, the MIS VDRE conferred calcitriol responsiveness to a heterologous promoter. In gel shift assays, VDR and retinoid X receptor bound to the MIS VDRE and the binding was increased by calcitriol. Chromatin immunoprecipitation assays showed that VDR and retinoid X receptor were present on the MIS promoter in prostate cancer cells. In conclusion, we demonstrated that MIS is a target of calcitriol action. MIS is up-regulated by calcitriol via a functional VDRE that binds the VDR. Up-regulation of MIS by calcitriol may be an important component of the antiproliferative actions of calcitriol in some cancers.

Genetic Defects of the 1,25-Dihydroxyvitamin D3 Receptor

Target organ resistance to steroid hormone action is known to produce clinical disorders ranging from testicular feminization in the case of androgen resistance to hypocalcemic vitamin D-resistant ricets (HVDRR) in the case of 1,25-dihydroxyvitamin D3. The etiologic basis of these disorders is thought to be genetic mutations in the gene encoding receptors for these hormones. We investigated this possibility by analyzing the vitamin D receptor (VDR) protein, mRNA, and DNA from patients with HVDRR. This autosomal recessive disease of children is characterized by early onset rickets, hypocalcemia, hyperparathyroidism, and elevated levels of 1,25-(OH)2D3. Cells from patients fall into three general classes of molecular defects: (i) decreased or absent hormone binding; (ii) decreased affinity of VDR for DNA, or; (iii) defective nuclear translocation or retention. Analysis of the DNA and/or mRNA from these cells has identified missense mutations in the DNA binding (zinc finger) domain and a nonsense mutation in the steroid binding domain of VDR. The mutations were individually recreated in wild type VDR and the expressed mutant protein behaved biochemically identically to the patient receptor. Further studies have shown that the receptor is unable to interact with the specific hormone response element (HRE) of the osteocalcin gene and activate appropriate transcription. Rapid diagnostic genotyping of these mutations is possible with either restriction digestion or allele-specific oligonucleotide hybridization. Analysis of these naturally occurring, disease producing mutations of a gene regulatory protein should provide insight into the key amino acid residues of the protein and the mechanism by which steroids modulate gene transcription.

The role of vitamin D receptor mutations in the development of alopecia.

Hereditary Vitamin D Resistant Rickets (HVDRR) is a rare disease caused by mutations in the vitamin D receptor (VDR). The consequence of defective VDR is the inability to absorb calcium normally in the intestine. This leads to a constellation of metabolic abnormalities including hypocalcemia, secondary hyperparathyroidism and hypophosphatemia that cause the development of rickets at an early age in affected children. An interesting additional abnormality is the presence of alopecia in some children depending on the nature of the VDR mutation. The data indicate that VDR mutations that cause defects in DNA binding, RXR heterodimerization or absence of the VDR cause alopecia while mutations that alter VDR affinity for 1,25(OH)(2)D(3) or disrupt coactivator interactions do not cause alopecia. The cumulative findings indicate that hair follicle cycling is dependent on unliganded actions of the VDR. Further research is ongoing to elucidate the role of the VDR in hair growth and differentiation.

Mutations in the vitamin D receptor and hereditary vitamin D-resistant rickets

Heterogeneous loss of function mutations in the vitamin D receptor (VDR) interfere with vitamin D signaling and cause hereditary vitamin D-resistant rickets (HVDRR). HVDRR is characterized by hypocalcemia, secondary hyperparathyroidism and severe early-onset rickets in infancy and is often associated with consanguinity. Affected children may also exhibit alopecia of the scalp and total body. The children usually fail to respond to treatment with calcitriol; in fact, their endogenous levels are often very elevated. Successful treatment requires reversal of hypocalcemia and secondary hyperparathyroidism and is usually accomplished by administration of high doses of calcium given either intravenously or sometimes orally to bypass the intestinal defect in VDR signaling.