Evaluation of a photolabile derivative of 1,25-dihydroxyvitamin D3 as a photoaffinity probe for 1,25-dihydroxyvitamin-D3 receptor in chick intestinal cytosol

We evaluated the viability of 1α,25-dihydroxyvitaminD₃-3β-[N-(4-azido-2-nitrophenyl)glycinate] (1,25-(OH)₂-D₃-ANG), an analog of 1α,25-dihydroxyvitamin D₃ (1,25-(OH)₂-D₃) as a photoaffinity probe for 1,25-(OH)₂-D₃ receptor in chick intestinal cytosol. A competitive-binding assay revealed that chick intestinal cytosolic 1,25-(OH)₂- D₃ receptor bound to 1,25-(OH)₂-D₃-ANG approximately 20-times less effectively than it did to 1,25-(OH)₂-D₃. Irradiation of 1,25-(OH)₂-D₃- ANG in the presence of chick intestinal cytosolic preparation significantly diminished subsequent binding to ³H-1,25-(OH)₂-D₃, suggesting that the photoaffinity analog was covalently attached to the receptor. Therefore the nitroarylazide derivative of 1,25-(OH)₂-D₃ may be a valuable photoaffinity probe for the characterization of the 1,25-(OH)₂-D₃ receptor.     

Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation

1,25-Dihydroxyvitamin D³ [1,25(OH)₂D₃] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum-free culture system, we have found that at physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)₂D₃. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)₂D₃ and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31-7549, had very little effect on the growth inhibition induced by a high dose (1 μM) of 1,25(OH)₂D₃. This lack of rapid signal transduction events was consistent with the inability of a short (4-hour) exposure to 1,25(OH)₂D₃ to initiate a complete growth-inhibitory response as measured using [³H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)₂D₃ are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)₂D₃ and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)₂D₃ receptor (VDR). © 1995 Wiley-Liss, Inc.     

1,25-Dihydroxy-vitamin-D3 enhances antiproliferative effect and transcription of TGF-beta1 on human keratinocytes in culture.

Both TGF-beta and 1,25-dihydroxy-vitamin-D3 (1,25(OH)2D3) have been reported to decrease the proliferation of normal human keratinocytes. The effect and expression of TGF-beta in keratinocytes treated with 1,25(OH)2D3 was investigated. Human keratinocytes were grown in the presence of various concentrations of TGF-beta and/or 1,25(OH)2D3 prior to enumeration. TGF-beta, alone, has a half maximal dose of inhibition (ED50) of approximately 750 pg/ml after seven days in culture in Keratinocyte Growth Medium (KGM; Clonetics) supplemented with 1.5 mM calcium. When 1,25(OH)2D3 (10(-7)M) was also added to cultures with various concentrations of TGF-beta, the ED50 shifted an average of 2-fold less. The presence of TGF-beta (10 pg/ml) augmented the potency of 1,25(OH)2D3 by at least 10-fold. In keratinocyte cultures, the antiproliferative effect of the two compounds together is synergistic. In keratinocytes grown for 1 week in the presence of 1,25(OH)2D3 at 10(-6)M, the TGF-beta 1 message increased approximately 5-fold. An increase is detected within 2 hours of exposure to 1,25(OH)2D3. There was only a 50% increase in the levels of TGF-beta 2 and no detection of TGF-beta 3. When keratinocyte cultures were treated with 1,25(OH)2D3 and neutralizing antibodies to TGF-beta, the induced-antiproliferative activity was blocked by more than 50%. The keratinocytes produced more active than latent TGF-beta after growth with high doses of 1,25(OH)2D3.     

Studies on calciferol metabolism: Metabolism of 25-hydroxy-Vitamin D3 by the chicken embryo

It is known that after birth of a vertebrate there is a requirement for the metabolism of Vitamin D₃ (cholecalciferol) to 1,25-(OH)₂-Vitamin D₃ to produce the hormonally active form essential for calcium homeostasis. However it is not known whether the enzymatic capability to produce 1,25-(OH)₂-D₃ only appears after birth or whether it is generated in the embryo. Presented in this paper are results of studies designed to measure the production and localization of 1,25-(OH)₂-D₃ in the embryo. It was found that the renal enzyme, 25-OH-cholecalciferol-1-hydroxylase, which is capable of producing 1,25-(OH)₂-D₃, is present as early as day 9 of incubation (12 days before hatch) in White Leghorn chicks. Further, the enzyme activity increases 6-fold to a maximal level which occurs on the day of hatching. 1,25-(OH)₂-D₃ was shown to be produced in vivo at day 17 and was found then in low levels in the embryonic intestine and kidney. Thus we have shown that 1,25-(OH)₂-D₃ is made by embryonic chick kidneys and is found in low levels in embryonic chick intestine and kidney significantly before hatch.     

Serum concentrations of 1,25-dihydroxyvitamin D3 in Platyrrhini and Catarrhini: A phylogenetic appraisal

We measured the serum concentration of 25-hydroxyvitamin D₃ (25-OH-D₃) and 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂-D₃) in 23 different Platyrrhines from four different genera and in 21 Catarrhines from six different genera in residence at the Los Angeles Zoo. The mean (±S.E.) serum concentration of 1,25-(OH)₂-D₃ was significantly greater in Platyrrhines (810 ± 119 pg/ml) than in Catarrhines (61 ± 5 pg/ml), suggesting that high circulating concentrations of the active vitamin D hormone were a characteristic of New World primates in both the Cebidae and Callitrichidae family. This increase in the serum concentration of 1,25-(OH)₂-D₃ is probably an adaptational response on the part of Platyrrhini to offset a relative decrease in the concentration of specific receptor for 1,25-(OH)₂-D₃ in target tissues for the hormone.     

Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: Structural and biochemical implications

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter ‘surface structure’ of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)₂D₃ containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)₂D₃. Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)₂D₃-1-BE), Cys288 (β-hairpin region: 1,25(OH)₂D₃-3-BE,) and Tyr295 (helix-6: 1,25(OH)₂D₃-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the β-hairpin region (modified by 1,25(OH)₂D₃-3-BE) is most important for growth inhibition by 1,25(OH)₂D₃, while helices 3 and 6 are less important for such activity.     

Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D₃, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D₃], and that 1,25(OH)₂D₃ endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH)₂D₃ is produced from 25(OH)D₃ by skin keratinocytes, 25(OH)[³H]D₃ was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH)₂D₃ was 1,25(OH)₂D₃. Most of the 1,25(OH)₂D₃ endogenously produced from 25(OH)D₃ remained within the cells. To examine the time course of 1,25(OH)₂D₃ production, the amount of 1,25(OH)[³H]D₃ was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D₃. These data indicate that keratinocytes rapidly convert 25(OH)D₃ to 1,25(OH)₂D₃ and that 1,25(OH)₂D₃ is not released into the medium. To determine whether endogenously produced 1,25(OH)₂D₃ is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH)₂D₃ and 25(OH)D₃ on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10^?8 M and 10^?7 M 1,25(OH)₂D₃ and to 80.5% and 23.9% by 10^?8 M and 10^?7 M 25(OH)D₃, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5·10^?7 M 1,25(OH)₂D₃, and to 11.8% by 2.5·10^?7 M 25(OH)D₃. These results clearly indicate that 25(OH)D₃ is as effective on human keratinocytes as 1,25(OH)₂D₃ in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH)₂D₃. The possibility that the effect of 25(OH)D₃ is mediated through binding to the 1,25(OH)₂D₃ receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D₃ for the 1,25(OH)₂D₃ receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH)₂D₃. Thus, these results suggest that 1,25(OH)₂D₃ endogenously produced in keratinocytes from 25(OH)D₃ is involved in the regulation of their growth and differentiation in vitro.     

Immunoreactive parathyroid hormone levels in platyrrhini and catarrhini: A comparative analysis with three different assays

Serum concentrations of the hormonal form of vitamin D₃—1,25-dihydroxy-vitamin D₃ [1,25-(OH)₂-D₃]—are elevated in many genera of platyrrhines when compared to catarrhines; this elevation is presumed to result from a decrease in the ability of the target cell receptor effectively to recognize 1,25-(OH)₂-D₃. The activity of the renal 25-hydroxyvitumin D₃-1α-hydroxylase, the mammalian enzyme which synthesizes the majority of the circulating 1,25-(OH)₂-D₃, is accelerated by parathyroid hormone (PTH). In order to determine whether the elevated serum concentrations of 1,25-(OH)₂-D₃ in platyrrhines were the result of relative hyperparathyroidism, we measured serum levels of immunoreactive parathyroid hormone (iPTH) in normocalcemic platyrrhines, catarrhines, and human subjects with assays that recognize different domains of the human PTH molecule. Antisera directed against the biologically active, aminoterminus of PTH yielded comparable mean values for iPTH among three test groups. The mean concentration of iPTH as assessed by a “proximal” midregion assay was significantly reduced in platyrrhine serum when compared to either human or catarrhine serum. A “distal” midregion assay yielded a reduced mean value for iPTH in both platyrrhine and catarrhine serum when compared to human serum. These data suggest that 1) high circulating levels of 1,25-(OH)₂-D₃ in New World primates are not the result of hyperparathyroidism; and 2) structural homology between human and primate PTH diminishes progressively as one moves toward the carboxyterminus of the molecule and is lost more rapidly in the platyrrhine than in the catarrhine hormone.     

Synthesis of and response to 1,25 dihydroxycholecalciferol by subpopulations of murine epidermal keratinocytes: Existence of a paracrine system for 1,25 dihydroxycholecalciferol

The epidermis is both a target tissue for and a source of 1,25 dihydroxycholecal-ciferol. The present study determines which of the epidermal cell populations synthesizes 1,25 dihydroxycholecalciferol and which responds to this hormone. Epidermal keratinocytes from new born rat epidermis were separated by unit gravity sedimentation into poorly differentiated cells, slow-cycling more differentiated cells, actively proliferating cells, and terminally differentiating subpopulations. The keratinocyte populations were characterized by cell size analysis, cell morphology, and DNA and RNA contents (acridine orange flow cytometry). 1,25(OH)₂D₃ synthesis was studied by measuring the conversion of [³H] 25(OH)D₃ to [³H] 1,25(OH)₂D₃. The purified product was tested for its ability to compete with synthetic [³H] 1,25(OH)₂D₃ for binding to chick intestinal cytosol. The responses of the keratinocyte subpopulations to exogeneous 1,25(OH)₂D₃ were evaluated by the increase in 25(OH)D₃-24 hydroxylase activity. Furthermore the expression of 1,25(OH)₂D₃ receptors (VDR) was examined in these cell populations. The results show that only the least differentiated cells produced 1,25(OH)₂D₃. In contrast, immunocytochemical detection of VDR, the VDR mRNA, and a 25(OH)D₃-24 hydroxylase response to 1,25(OH)₂D₃ were mainly found in the more differentiated cells. Thus, the ability of epidermis to synthesize 1,25(OH)₂D₃ and be simultaneously sensitive to it depends on the state of cell differentiation. This suggests that the mammalian epidermis contains a paracrine system in which the more differentiated keratinocytes are sensitive to the 1,25(OH)₂D₃ produced locally by neighboring immature ones. © 1994 wiley-Liss, Inc.     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.     

1,25 dihydroxyvitamin D3 enhances the calcium response of keratinocytes

The steroid hormone 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) regulates cell proliferation and differentiation. Intracellular calcium (Cai) concentrations play a crucial role in these events. From our previous studies, we have demonstrated a calcium receptor (CaR) in keratinocytes which appears to regulate the initial release of Cai from intracellular stores in response to extracellular calcium (Cao) and so is likely to participate in the differentiation process. In this study, we determined whether the ability of 1,25(OH)2D3 to enhance Ca++ -induced differentiation was mediated at least in part through changes in the CaR. Keratinocytes were grown in keratinocyte growth medium (KGM) with 0.03 mM, 0.1 mM, or 1.2 mM Ca and treated with 10(-8) M 1,25(OH)2D3 till harvest after 5, 7, 14, and 21 days. CaR mRNA levels were quantitated by polymerase chain reaction. The results were compared to the ability of 1,25(OH)2D3 to enhance calcium-stimulated increases in Cai. In cells grown in 0.03 mM Ca, the CaR mRNA levels decreased with time. 1,25(OH)2D3 stimulated the levels at 5 days and prevented the falloff over the subsequent 16 days. On the other hand, in cells grown in 0.1 or 1.2 mM Ca, the message levels remained high, and 1,25(OH)2D3 had no further effect. To study the functional relationship, we harvested cells after 5 and 7 days in culture following a 24 h treatment with 1,25(OH)2D3 or vehicle to measure the Cai response to 2 mM Cao. The preconfluent cells grown in 0.03 mM Ca showed a nearly twofold increase in the Cai response to Cao when pretreated with 1,25(OH)2D3, whereas the confluent cells and those grown in 1.2 mM Ca showed no enhancement by 1,25(OH)2D3. Studies with 45Ca influx into keratinocytes revealed that 1,25(OH)2D3 enhanced the influx in preconfluent and confluent cells when grown in KGM containing 0.03 mM Ca but not in cells grown in 1.2 mM calcium. We conclude that 1,25(OH)2D3 maintains the CaR mRNA levels in cells grown in 0.03 mM Ca, thus maintaining their responsiveness to Cao and so ensuring their ability to differentiate in response to the calcium signal.     

Early effect of 25-hydroxycholecalciferol (25-OH-D3) and 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) on the ability of parathyroid hormone (PTH) to elevate cyclic AMP of intact bone cells.

25-OH-D₃ and 1,25-(OH)₂-D₃ had no effects by themselves on the cyclic AMP levels of isolated bone cells but enhanced the stimulation seen following an exposure with submaximal concentrations of PTH for as little as 2 minutes. Preincubation with the 25-OH-D₃ or 1,25-(OH)₂-D₃ resulted in a time dependent decrease in the enhancement of PTH response over a 1 hr period. It is, therefore, suggested that cyclic AMP may be involved in some aspects of the action of vitamin D₃ derivatives on bone cells.     

The MRNA expression of the human 1,25-dihydroxyvitamin D3 receptor and the c-myc protooncogene in cultured human keratinocytes

Summary The human vitamin D receptor mRNA expression in preconfluent human cultured keratinocytes was upregulated by treatment of these cells with 10^−8 M 1,25(OH)₂D₃ for 24 hours. Additionally, human c-myc mRNA expression was decreased in a dose dependent manner by 1,25(OH)₂D₃ in both preconfluent and confluent cultured human keratinocytes.     

Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes.

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D3], and that 1,25(OH)2D3 endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH)2D3 is produced from 25(OH)D3 by skin keratinocytes, 25(OH)[3H]D3 was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH)D3 was 1,25(OH)2D3. Most of the 1,25(OH)2D3 endogenously produced from 25(OH)D3 remained within the cells. To examine the time course of 1,25(OH)2D3 production, the amount of 1,25(OH)[3H]D3 was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D3. These data indicate that keratinocytes rapidly convert 25(OH)D3 to 1,25(OH)2D3 and that 1,25(OH)2D3 is not released into the medium. To determine whether endogenously produced 1,25(OH)2D3 is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH)2D3 and 25(OH)D3 on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10(-8) M and 10(-7) M 1,25(OH)2D3 and to 80.5% and 23.9% by 10(-8) M and 10(-7) M 25(OH)D3, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5.10(-7) M 1,25(OH)2D3, and to 11.8% by 2.5.10(-7) M 25(OH)D3. These results clearly indicate that 25(OH)D3 is as effective on human keratinocytes as 1,25(OH)2D3 in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH)2D3. The possibility that the effect of 25(OH)D3 is mediated through binding to the 1,25(OH)2D3 receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D3 for the 1,25(OH)2D3 receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH)2D3. Thus, these results suggest that 1,25(OH)2D3 endogenously produced in keratinocytes from 25(OH)D3 is involved in the regulation of their growth and differentiation in vitro.     

1,25-dihydroxyvitamin D3 Activates MMP13 Gene Expression in Chondrocytes through p38 MARK Pathway

Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)₂D₃ in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)₂D₃ on chondrocytes in OA remains obscure. Effect of 1,25-(OH)₂D₃ on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)₂D₃ activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)₂D₃-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)₂D₃ on MMP13 expression, transfection assays were used to show that 1,25-(OH)₂D₃ activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)₂D₃ in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)₂D₃ activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)₂D₃ to potentiate MMP13 expression might facilitate cartilage erosion at the site of inflammatory arthritis.     

Targeted disruption of the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in ras-transformed keratinocytes demonstrates that locally produced 1alpha,25-dihydroxyvitamin D3 suppresses growth and induces differentiation in an autocrine fashion

It has been previously shown that keratinocytes express a high level of 25-hydroxyvitamin D(3) (25-OHD(3)) 1alpha-hydroxylase (1alpha-hydroxylase). 1alpha-Hydroxylase catalyzes the conversion of 25-OHD(3) to 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3) is both antiproliferative (i.e., suppresses cell growth) and prodifferentiative (i.e., induces cell differentiation) in many cell types. We hypothesized that local production of 1,25(OH)(2)D(3) by keratinocytes may suppress their growth and induce their differentiation in an autocrine fashion. To test this hypothesis, we inactivated both 1alpha-hydroxylase alleles in a ras-transformed keratinocyte cell line, HPK1Aras, which typically produces squamous carcinoma in nude mice. To inactivate 1alpha-hydroxylase expression by HPK1Aras cells, we disrupted both alleles of the 1alpha-hydroxylase gene by homologous recombination. Lack of expression and activity of 1alpha-hydroxylase was confirmed by Northern blot analysis and detected conversion of 25-OHD(3) to 1,25(OH)(2)D(3). We then examined the effect of substrate 25-OHD(3) on parameters of growth and differentiation in the double knockout cell line as compared to wild-type HPK1Aras cells in vitro. It was found that 1alpha-hydroxylase inactivation blocked the antiproliferative and prodifferentiative effect of 25-OHD(3). These in vitro effects were further analyzed in vivo by injecting knockout or control cells subcutaneously in severely compromised immunodeficient mice. Tumor growth was accelerated and differentiation was inhibited in mice given injections of knockout cells as compared to control cells in the presence of substrate 25-OHD(3). Our results demonstrate, for the first time, that 1alpha-hydroxylase expression by keratinocytes plays an important role in autocrine growth and differentiation of these cells, and suggest that expression of this enzyme may modulate tumor growth in squamous carcinomas.     

All-trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25-Dihydroxyvitamin D3 in normal human keratinocytes

1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)₂D₃ and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)₂D₃ and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)₂D₃ and to all but the highest concentration (10⁻⁶ M) of RA. 1,25(OH)₂D₃ exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10⁻⁹ M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)₂D₃. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)₂D₃ to raise INV levels, possibly by inhibiting 1,25(OH)₂D₃-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)₂D₃, but assessment of a single marker, such as INV protein, may be misleading. J. Cell. Physiol. 174:1–8, 1998. © 1998 Wiley-Liss, Inc.     

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)₂D₃ traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)₂D₃ called 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D₃-MARRS expression modulates 1,25(OH)₂D₃ activity in breast cancer cells.Relative levels of 1,25D₃-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D₃-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)₂D₃ in MCF-7 cells, a ribozyme construct designed to knock down 1,25D₃-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D₃-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)₂D₃ ( IC₅₀ 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D₃-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)₂D₃. Knockdown of 1,25D₃-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D₃-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)₂D₃ in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D₃-MARRS expression or activity as anticancer agents.     

Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3).

1alpha,25-Dihydroxyvitamin D(3)-3-bromoacetate (1, 25(OH)(2)D(3)-3-BE), an affinity labeling analog of 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), displayed stronger antiproliferative activities than 1,25(OH)(2)D(3) at 10(-10)-10(-6) M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10(-6) M 1,25(OH)(2)D(3), followed by treatment with various doses of 1,25(OH)(2)D(3)-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [(14)C]1, 25(OH)(2)D(3)-3-BE covalently labeled human recombinant 1alpha, 25-dihydroxyvitamin D(3) receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)(2)D(3), by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [(14)C]1,25(OH)(2)D(3)-3-BE in CHK extract, indicating that VDR is the main target of [(14)C]1, 25(OH)(2)D(3)-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)(2)D(3)-controlled genes. Furthermore, we observed that 1,25(OH)(2)D(3)-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)(2)D(3) in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1, 25(OH)(2)D(3)-3-BE was found to be more potent in inducing 1alpha, 25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1, 25(OH)(2)D(3)-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1, 25(OH)(2)D(3)-VDR complex. This resulted in an enhanced stability of the 1,25(OH)(2)D(3)-3-BE/VDR-VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)(2)D(3)-3-BE in keratinocytes.