Macrophage Inflammatory Proteins (MIPs) Contribute to Malignant Potential of Colorectal Polyps and Modulate Likelihood of Cancerization Associated with Standard Risk Factors.

Better understanding of molecular changes leading to neoplastic transformation is prerequisite to optimize risk assessment and chemopreventive and surveillance strategies. Data on macrophage inflammatory proteins (MIPs) in colorectal carcinogenesis are scanty and their clinical relevance remains unknown. Therefore, transcript and protein expression of CCL3, CCL4, CXCL2, and CCL19 were determined in 173 and 62 patients, respectively, using RT-qPCR and immunohistochemistry with reference to polyps' characteristics. The likelihood of malignancy was modeled using probit regression. With the increasing malignancy potential of hyperplastic-tubular-tubulo-villous-villous polyps, the expression of CCL3, CCL4, and CCL19 in lesions decreased. CCL19 expression decreased also in normal mucosa while that of CXCL2 increased. Likewise, lesion CCL3 and lesion and normal mucosa CCL19 decreased and normal CXCL2 increased along the hyperplasia-low-high dysplasia grade. The bigger the lesion, the lower CCL3 and higher CXCL2 in normal mucosa. Singular polyps had higher CCL3, CCL4, and CCL19 levels in normal mucosa. CCL3, CCL4 and CXCL2 modulated the likelihood of malignancy associated with traditional risk factors. There was no correlation between the protein and mRNA expression of CCL3 and CCL19. In summary, the polyp-adjacent mucosa contributes to gaining potential for malignancy by polyps. MIPs may help in specifying cancerization probability estimated based on standard risk factors.

Transcriptome Analysis Reveals Intrinsic Proinflammatory Signaling in Healthy African American Skin.

Differences in the morphology and physiology of darkly pigmented skin compared with those of lightly pigmented skin are well-recognized. There are also disparities in the prevalence and clinical features for many inflammatory skin diseases, including atopic dermatitis and psoriasis; however, the underlying mechanisms are largely unknown. We compared the baseline gene expression in full-thickness skin biopsies from healthy individuals self-reporting as African American (AA) or as White non-Hispanic (WNH). Extensively validated RNA-sequencing analysis identified 570 differentially expressed genes in AA skin, including Igs and their receptors such as FCER1G; proinflammatory genes such as TNFα and IL32; and epidermal differentiation cluster and keratin genes. Differentially expressed genes were functionally enriched for inflammatory responses, keratinization, and cornified envelope formation. RNA-sequencing analysis of three-dimensional human skin equivalents made from AA and WNH primary keratinocytes revealed 360 differentially expressed genes (some shared with skin) that were enriched by similar functions. AA human skin equivalents appeared more responsive to TNF-α proinflammatory effects. Finally, AA-specific differentially expressed genes in the skin and human skin equivalents significantly overlapped with molecular signatures of skin in patients with atopic dermatitis and psoriasis. Overall, these findings suggest the existence of intrinsic proinflammatory circuits in AA keratinocytes/skin that may account for disease disparities and will help to build a foundation for the development of targeted skin disease prevention.

Early Stress-Response Gene REDD1 Controls Oxazolone-Induced Allergic Contact Dermatitis.

REDD1 is an energy sensor and stress-induced mTOR inhibitor. Recently, its novel role in linking metabolism and inflammation/immune responses has emerged. In this study, we assessed the role of REDD1 in murine oxazolone-induced allergic contact dermatitis (ACD), a T cell-dependent model with features of human ACD. A variety of immune indices, including edema, cellular infiltration, inflammatory gene expression, and glucocorticoid response, were compared in Redd1 knockout (KO) and isogenic (C57BL/6 × 129)F1 wild-type mice after sensitization and subsequent ear challenge with oxazolone. Despite relatively normal thymic profiles and similar T cell populations in the lymph nodes of naive Redd1 KO mice, early T cell expansion and cytokine production were profoundly impaired after sensitization. Surprisingly, higher steady-state populations of CD4+ and CD8+ T cells, as well as macrophages (CD45+/Ly-6G-/CD11b+), dendritic cells (CD45+/Ly-6G-/CD11c+), neutrophils (CD45+/Ly-6G+/CD11b+), and innate lymphoid cells (CD45+/Lineage-/IL-7Ra+/ST2+/c-Kit+), were observed in the ears of naive Redd1 KO mice. Upon challenge, ear edema, T cell, macrophage, neutrophil, and dendritic cell infiltration into the ear was significantly reduced in Redd1 KO animals. Accordingly, we observed significantly lower induction of IFN-γ, IL-4, and other cytokines as well as proinflammatory factors, including TSLP, IL-33, IL-1ß, IL-6, and TNF-α, in challenged ears of Redd1 KO mice. The response to glucocorticoid treatment was also diminished. Taken together, these data establish REDD1 as an essential immune modulator that influences both the initiation of ACD disease, by driving naive T cell activation, and the effector phase, by promoting immune cell trafficking in T cell-mediated skin inflammation.

A Novel Approach to Safer Glucocorticoid Receptor-Targeted Anti-lymphoma Therapy via REDD1 (Regulated in Development and DNA Damage 1) Inhibition.

Glucocorticoids are widely used for therapy of hematologic malignancies. Unfortunately, chronic treatment with glucocorticoids commonly leads to adverse effects including skin and muscle atrophy and osteoporosis. We found recently that REDD1 (regulated in development and DNA damage 1) plays central role in steroid atrophy. Here, we tested whether REDD1 suppression makes glucocorticoid-based therapy of blood cancer safer. Unexpectedly, approximately 50% of top putative REDD1 inhibitors selected by bioinformatics screening of Library of Integrated Network-Based Cellular Signatures database (LINCS) were PI3K/Akt/mTOR inhibitors. We selected Wortmannin, LY294002, and AZD8055 for our studies and showed that they blocked basal and glucocorticoid-induced REDD1 expression. Moreover, all PI3K/mTOR/Akt inhibitors modified glucocorticoid receptor function shifting it toward therapeutically important transrepression. PI3K/Akt/mTOR inhibitors enhanced anti-lymphoma effects of Dexamethasone in vitro and in vivo, in lymphoma xenograft model. The therapeutic effects of PI3K inhibitor+Dexamethasone combinations ranged from cooperative to synergistic, especially in case of LY294002 and Rapamycin, used as a previously characterized reference REDD1 inhibitor. We found that coadministration of LY294002 or Rapamycin with Dexamethasone protected skin against Dexamethasone-induced atrophy, and normalized RANKL/OPG ratio indicating a reduction of Dexamethasone-induced osteoporosis. Together, our results provide foundation for further development of safer and more effective glucocorticoid-based combination therapy of hematologic malignancies using PI3K/Akt/mTOR inhibitors.

Regulated in Development and DNA Damage Responses 1 Prevents Dermal Adipocyte Differentiation and Is Required for Hair Cycle-Dependent Dermal Adipose Expansion.

Dermal white adipose tissue (dWAT) expansion is associated with important homeostatic and pathologic processes in skin. Even though mTOR/protein kinase B signaling is important for adipogenesis, the role of regulated development of DNA damage responses 1 (REDD1), a negative regulator of mTOR/protein kinase B, is poorly understood. Loss of REDD1 in mice resulted in reduction of body mass, total fat, size of gonadal white adipose tissue, and interscapular brown adipose tissue. Inguinal subcutaneous white adipose tissue and dWAT in REDD1 knockouts were expanded compared with wild type mice. Size and number of mature adipocytes in dWAT were also increased in adult REDD1 knockouts. This dWAT phenotype was established around postnatal day 18 and did not depend on the hair growth cycle. Numbers of adipocyte precursor cells were lower in REDD1 knockout skin. In vitro analysis revealed increased differentiation of skin-derived REDD1 knockout adipocyte precursor cells as indicated by higher lipid accumulation and increased adipogenic marker expression. 3T3L1 cells overexpressing REDD1 had decreased sensitivity to differentiation. Overall, our findings indicate that REDD1 silencing induced expansion of dWAT through hypertrophy and hyperplasia. This REDD1-dependent mechanism of adipogenesis could be used to preferentially target skin-associated adipose tissue for therapeutic purposes.

Lipidomic analysis of human primary hepatocytes following LXR activation with GW3965 identifies AGXT2L1 as a main target associated to changes in phosphatidylethanolamine.

Liver X receptor (LXR) agonists have the potential to alleviate obesity related diseases, particularly atherosclerosis. However, LXRs are transcriptional regulators that induce de novo lipogenesis and lipid accumulation in hepatocytes which represents a serious adverse effect. In this work, we sought to characterize the LXR agonist GW3965 effects on fatty acid (FA) and phospholipid (PL) remodelling and the correlation with gene expression in order to better understand the underlying effects leading to hepatic pathology upon LXR activation. Human primary hepatocytes treated for 48 h with GW3965 were analysed for changes in lipid metabolism gene expression by qPCR, variations in the FA profile was evaluated by GC-FID and in PL profiles using thin layer chromatography, ESI-MS and MS/MS analysis. Changes in cell membrane biochemical properties were studied using bilayer models generated with CHARMM-GUI. ELOLV6 and SCD1 mRNA increase was consistent with higher C16:1 and C18:1n9 at the expense of C16:0 and C18:0. The reduction of C18:2n6 and increase in C20:2n6 was in agreement with ELOVL5 upregulation. Phosphatydilethanolamine (PE) levels tended to decrease and phosphatidylinositol to increase; although differences did not reach significance, they correlated with changes in AGXT2L1, CDS1 and LPIN1 mRNA levels that were increased. The overall effect of GW3965 on PEs molecular profiles was an increase of long-chain polyunsaturated FA chains and a decrease of C16/C18 saturated and monounsaturated FAs chains. Additionally, PC (32:1) and PC (34:2) were decreased, and PC (36:1) and PC (34:1) were increased. AGXT2L1 is an enzyme with strict substrate specificity for phosphoethanolamine, which is converted into ammonia in GW3965-treated hepatocytes and could explain the PE reduction. In summary, LXR activation by GW3965 targets PE biosynthesis and FA elongation/desaturation, which tends to decrease PE in relation to total PL levels, and remodelling of PC and PE molecular species. We identified the human AGXT2L1 gene as induced by LXR activation by both synthetic and endogenous agonist treatment. The increase in acetaldehyde-induced oxidative stress, and in the lipid species identified have the potential to enhance the inflammatory process and impair membrane function. Future studies should focus on inhibition of AGXT2L1 activity with the aim of reverting the steatosis induced by LXR activation.

Transcriptomic Network Interactions in Human Skin Treated with Topical Glucocorticoid Clobetasol Propionate.

Glucocorticoids are the most frequently used anti-inflammatory drugs in dermatology. However, the molecular signature of glucocorticoids and their receptor in human skin is largely unknown. Our validated bioinformatics analysis of human skin transcriptome induced by topical glucocorticoid clobetasol propionate (CBP) in healthy volunteers identified numerous unreported glucocorticoid-responsive genes, including over a thousand noncoding RNAs. We observed sexual and racial dimorphism in the CBP response including a shift toward IFN-α/IFN-γ and IL-6/Jak/Signal transducer and activator of transcription (STAT) 3 signaling in female skin; and a larger response to CBP in African-American skin. Weighted gene coexpression network analysis unveiled a dense skin network of 41 transcription factors including circadian Kruppel-like factor 9 (KLF9), and ∼260 of their target genes enriched for functional pathways representative of the entire CBP transcriptome. Using keratinocytes with Kruppel-like factor 9 knockdown, we revealed a feedforward loop in glucocorticoid receptor signaling, previously unreported. Interestingly, many of the CBP-regulated transcription factors were involved in the control of development, metabolism, circadian clock; and 80% of them were associated with skin aging showing similarities between glucocorticoid-treated and aged skin. Overall, these findings indicate that glucocorticoid receptor acts as an important regulator of gene expression in skin-both at the transcriptional and posttranscriptional level-via multiple mechanisms including regulation of noncoding RNAs and multiple core transcription factors.

Bacteriophages engineered to display foreign peptides may become short-circulating phages.

Bacteriophages draw scientific attention in medicine and biotechnology, including phage engineering, widely used to shape biological properties of bacteriophages. We developed engineered T4-derived bacteriophages presenting seven types of tissue-homing peptides. We evaluated phage accumulation in targeted tissues, spleen, liver and phage circulation in blood (in mice). Contrary to expectations, accumulation of engineered bacteriophages in targeted organs was not observed, but instead, three engineered phages achieved tissue titres up to 2 orders of magnitude lower than unmodified T4. This correlated with impaired survival of these phages in the circulation. Thus, engineering of T4 phage resulted in the short-circulating phage phenotype. We found that the complement system inactivated engineered phages significantly more strongly than unmodified T4, while no significant differences in phages' susceptibility to phagocytosis or immunogenicity were found. The short-circulating phage phenotype of the engineered phages suggests that natural phages, at least those propagating on commensal bacteria of animals and humans, are naturally optimized to escape rapid neutralization by the immune system. In this way, phages remain active for longer when inside mammalian bodies, thus increasing their chance of propagating on commensal bacteria. The effect of phage engineering on phage pharmacokinetics should be considered in phage design for medical purposes.

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles.

The most common method for phage quantitation is the plaque assay, which relies on phage ability to infect bacteria. However, non-infective phage particles may preserve other biological properties; specifically, they may enter interactions with the immune system of animals and humans. Here, we demonstrate real-time quantitative polymerase chain reaction (qPCR) detection of bacteriophages as an alternative to the plaque assay. The closely related staphylococcal bacteriophages A3R and 676Z and the coliphage T4 were used as model phages. They were tested in vivo in mice, ex vivo in human sera, and on plastic surfaces designed for ELISAs. T4 phage was injected intravenously into pre-immunized mice. The phage was completely neutralized by specific antibodies within 5 h (0 pfu/ml of serum, as determined by the plaque assay), but it was still detected by qPCR in the amount of approximately 107 pfu/ml of serum. This demonstrates a substantial timelapse between "microbiological disappearance" and true clearance of phage particles from the circulation. In human sera ex vivo, qPCR was also able to detect neutralized phage particles that were not detected by the standard plaque assay. The investigated bacteriophages differed considerably in their ability to immobilize on plastic surfaces: this difference was greater than one order of magnitude, as shown by qPCR of phage recovered from plastic plates. The ELISA did not detect differences in phage binding to plates. Major limitations of qPCR are possible inhibitors of the PCR reaction or free phage DNA, which need to be considered in procedures of phage sample preparation for qPCR testing. We propose that phage pharmacokinetic and pharmacodynamic studies should not rely merely on detection of antibacterial activity of a phage. Real-time qPCR can be an alternative for phage detection, especially in immunological studies of bacteriophages. It can also be useful for studies of phage-based drug nanocarriers or biosensors.

T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo.

Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo.

Immune response of macrophages induced by Porphyromonas gingivalis requires HmuY protein.

The main etiologic agent and a key pathogen responsible for initiation and progression of chronic periodontitis is Porphyromonas gingivalis. We examined the role of P. gingivalis, with particular interest to HmuY protein, in expression of genes involved in Toll-like receptor (TLR)-induced signaling pathways using cell-based infection model. U937 and THP-1 cells differentiated toward macrophages by PMA treatment responded to P. gingivalis-caused infection in slightly different gene expression pattern, mainly by higher expression of genes encoding NF-κB, TLR7, TLR2, TLR8, pro-inflammatory cytokines (IL-1ß, IL-6, TNFα), anti-inflammatory cytokine (IL-10), and chemokines (CCL3L1, CCL4, CXCL10, CXCL11, PTX3). P. gingivalis lacking functional hmuY gene stimulates immune response of macrophages, albeit in a different manner as compared with the wild-type strain, mainly by lower expression of genes encoding NF-κB, IL-1ß, IL-10, CD80, PTX3, and CCL31L. The purified HmuY protein alone induced expression of genes encoding IL-6, IL-10, TNFα, CCL3L1, and CCL4. We conclude that macrophages respond to P. gingivalis infection mostly by TLR7-induced pathway(s). Moreover, P. gingivalis HmuY is one of important virulence factors, which allows P. gingivalis for in vivo growth in the heme-limited host environment, resulting in efficient immune response of macrophages.

Mammalian Host-Versus-Phage immune response determines phage fate in vivo.

Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy.

Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood.

A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.

Selective Activator of the Glucocorticoid Receptor Compound A Dissociates Therapeutic and Atrophogenic Effects of Glucocorticoid Receptor Signaling in Skin.

BACKGROUND: Glucocorticoids are effective anti-inflammatory drugs widely used in dermatology and for the treatment of blood cancer patients. Unfortunately, chronic treatment with glucocorticoids results in serious metabolic and atrophogenic adverse effects including skin atrophy. Glucocorticoids act via the glucocorticoid receptor (GR), a transcription factor that causes either gene transactivation (TA) or transrepression (TR). Compound A (CpdA), a novel non-steroidal GR ligand, does not promote GR dimerization and TA, retains anti-inflammatory potential but induces fewer metabolic side effects compared to classical glucocorticoids when used systemically. As topical effects of CpdA have not been well studied, this work goal was to compare the anti-inflammatory and side effects of topical CpdA and glucocorticoids and to assess their effect on GR TA and TR in keratinocytes. METHODS: We used murine immortalized keratinocytes and F1 C57BlxDBA mice. Effect of glucocorticoid fluocinolone acetonide (FA) and CpdA on gene expression in keratinocytes in vitro and in vivo was evaluated by reverse transcription-PCR. The anti-inflammatory effects were assessed in the model of tumor promoter 12-O-tertradecanoyl-acetate (TPA)-induced dermatitis and in croton oil-induced ear edema test. Skin atrophy was assessed by analysis of epidermal thickness, keratinocyte proliferation, subcutaneous adipose hypoplasia, and dermal changes after chronic treatment with FA and CpdA. RESULTS: In mouse keratinocytes in vitro and in vivo, CpdA did not activate GR-dependent genes but mimicked closely the inhibitory effect of glucocorticoid FA on the expression of inflammatory cytokines and matrix metalloproteinases. When applied topically, CpdA inhibited TPA-induced skin inflammation and hyperplasia. Unlike glucocorticoids, CpdA itself did not induce skin atrophy which correlated with lack of induction of atrophogene regulated in development and DNA damage response 1 (REDD1) causatively involved in skin and muscle steroid-induced atrophy. CONCLUSIONS: Overall, our results suggest that CpdA and its derivatives represent novel promising class of anti-inflammatory compounds with reduced topical side effects.

Studies on the mechanisms of superagonistic pro-differentiating activities of side-chain modified analogs of vitamin D2.

1,25-Dihydroxyvitamin D3 (1,25D) is implicated in many cellular functions including cell proliferation and differentiation, thus, exerting potential antitumor effects. A major limitation for therapeutic use of 1,25D are its potent calcemic and phosphatemic activities. Therefore, synthetic analogs of 1,25D for use in anticancer therapy should retain cell differentiating potential, with calcemic activity being reduced. Previously, we described pro-differentiating effects of vitamin D2 analogs with extended and branched side-chains. Analogs with side-chains extended by a pair of one (PRI-1906) or two carbon units (PRI-1907) displayed elevated cell-differentiating activity towards some acute leukemia cell lines (AML) in comparison to 1,25D. In this study, the potential mechanism of this superagonistic activity of the analogs was addressed. At first, possible differences in the expression of CYP24A1, a major catabolizing enzyme for vitamin D compounds and resulting differences in the degradation of analogs were investigated. In addition, interactions of the analogs with vitamin D receptor (VDR) and resulting activation of CCAAT-enhancer-binding protein ß (C/EBPß) were studied. The results obtained show that superagonistic pro-differentiating activities of analogs PRI-1906 and PRI-1907 do not seem to be caused by their altered catabolism, but most probably by altered interactions with VDR and resulting downstream proteins.

The effect of differentiation on 1,25 dihydroxyvitamin D-mediated gene expression in the enterocyte-like cell line, Caco-2.

We examined 1,25 dihydroxyvitamin D (1,25(OH)(2)D(3))-induced expression of 25-hydroxyvitamin D(3) 24-hydroxylase (CYP24) and apical calcium channel (TRPV6) mRNA levels in 2-, 9-, and 15-day cultures Caco-2 cells that model proliferating, post-proliferative, and differentiated enterocytes. 1,25(OH)(2)D(3)-induced (10 nM, 8 h) CYP24 and TRPV6 mRNA levels were significantly greater in differentiated and post-proliferative than proliferating Caco-2 cells (>16X and >3X, respectively). Neither CYP24 mRNA half-life nor induction of a -298 bp rat CYP24 promoter-luciferase reporter construct (10 nM 1,25(OH)(2)D(3), 24 h) were different between proliferating and post-proliferating Caco-2 cells. We next tested whether the blunted response of natural genes to 1,25(OH)(2)D(3) in proliferating Caco-2 cells is due to altered chromatin remodeling. VDR and coactivator protein levels do not increase with differentiation but the level of the co-repressor Alien falls by 50% with differentiation. Over-expression of Alien reduced 1,25(OH)(2)D(3)-induced activity of a minimal VDRE containing promoter-luciferase construct by more than 60% in differentiated Caco-2 cells while siRNA knockdown of Alien in proliferating Caco-2 cells increased 1,25(OH)(2)D(3)-induced CYP24 mRNA level by 40%. These observations suggest that Alien is a regulator of VDR-mediated gene transcription in Caco-2 cells. In addition, we found that 1,25(OH)(2)D(3)-induced association of VDR with chromatin and with the CYP24 promoter was lower in proliferating cells. This suggests that decreased recruitment of VDR to vitamin D response elements also contributes to the blunted transcriptional responsiveness to 1,25(OH)(2)D(3) in proliferating Caco-2 cells.

1,25-dihydroxyvitamin D3 suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin gene promoter.

We have shown that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from -2,725 to -2,647 (distal fragment) and from -117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)(2)D(3) inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)(2)D(3)-induced suppression. EMSA revealed that 1,25(OH)(2)D(3) markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)(2)D(3) suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.

Nucleo-cytoplasmic cycling of the vitamin D receptor in the enterocyte-like cell line, Caco-2.

We examined the effects of 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on the distribution and mobility of the vitamin D receptor (VDR) in the enterocyte-like Caco-2 cell. Confocal microscopy showed that a green fluorescent protein-vitamin D receptor (GFP-VDR) fusion protein is predominantly nuclear (58%) and it does not associate with the apical or basolateral membrane of proliferating or polarized, differentiated cells. In contrast to the previously studied cell types, neither endogenous VDR nor GFP-VDR levels accumulate in the nucleus following 1,25(OH)(2)D(3) treatment (100 nM, 30 min). However, in nuclear photobleaching experiments nuclear GFP-VDR import was significantly increased by 1,25(OH)(2)D(3) during both an early (0-5 min) and later (30-35 min) period (20% per 5 min). Compared to the natural ligand, nuclear import of GFP-VDR was 60% lower in cells treated with the 1,25(OH)(2)D(3) analog, 1-alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228, 5 min, 100 nM). Downstream events like ligand-induced association of VDR with chromatin at 1 h and the accumulation of CYP24 mRNA were significantly lower in Ro-26-9228 treated cells compared to 1,25(OH)(2)D(3) (60 and 95% lower, respectively). Collectively our data are consistent with a role for ligand-induced nuclear VDR import in receptor activation. In addition, ligand-dependent VDR nuclear import appears to be balanced by export, thus accounting for the lack of nuclear VDR accumulation even when VDR import is significantly elevated.

Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1alpha.

Calbindin D9k (CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH)2 vitamin D (<2-fold) in the Caco-2 clone TC7. Deletion-mutation studies revealed that conserved promoter sequences for cdx-2 (at -3158 bp) and hepatocyte nuclear factor (HNF)-1 (at -3131 and at -98 bp) combined to control CaBP expression during differentiation. Other putative response elements were not important for CaBP regulation in TC7 cells (CCAAT enhancer binding protein, pancreatic duodenal homebox-1 (pdx-1), a proximal cdx-2 element). Mutation of the distal HNF-1 site had the greatest impact on CaBP gene expression through disruption of HNF-1alpha binding; both basal and differentiation-mediated CaBP expression was reduced by 80%. In contrast, mutation of the distal cdx-2 element reduced only basal CaBP expression. Whereas a 60% reduction of CaBP mRNA in the duodenum of HNF-1alpha null mice confirmed the physiological importance of HNF-1alpha for CaBP gene regulation, additional studies showed that maximal CaBP expression requires the presence of both HNF-1alpha and cdx-2. Our data suggest that cdx-2 is a permissive factor that influences basal CaBP expression in enterocytes and that HNF-1alpha modulates CaBP gene expression during cellular differentiation.