Antifibrotic vitamin D analogs

Chronic kidney disease is associated with progressive kidney fibrosis, which disrupts normal kidney function. There is a great need for treatments to reduce renal fibrosis. In this issue of the JCI, Ito and colleagues report the development of synthetic ligands of the vitamin D receptor that target the TGF-β–SMAD signaling pathway, which is known to regulate fibrosis-associated gene expression, without inducing VDR-associated genes. These ligands ameliorated renal fibrosis in two different mouse models. This study justifies further investigation of these and related compounds for treatment of humans with chronic kidney disease or other diseases characterized by fibrosis.     

Vitamin D3 attenuates Th2 responses to Aspergillus fumigatus mounted by CD4+ T cells from cystic fibrosis patients with allergic bronchopulmonary aspergillosis

Allergic bronchopulmonary aspergillosis (ABPA) is caused by a dominant Th2 immune response to antigens derived from the opportunistic mold Aspergillus, most commonly Aspergillus fumigatus. It occurs in 4%–15% of patients with cystic fibrosis (CF); however, not all patients with CF infected with A. fumigatus develop ABPA. Therefore, we compared cohorts of A. fumigatus–colonized CF patients with and without ABPA to identify factors mediating tolerance versus sensitization. We found that the costimulatory molecule OX40 ligand (OX40L) was critical in driving Th2 responses to A. fumigatus in peripheral CD4⁺ T cells isolated from patients with ABPA. In contrast, CD4⁺ T cells from the non-ABPA cohort did not mount enhanced Th2 responses in vitro and contained a higher frequency of TGF-β–expressing regulatory T cells. Heightened Th2 reactivity in the ABPA cohort correlated with lower mean serum vitamin D levels. Further, in vitro addition of 1,25 OH-vitamin D₃ substantially reduced DC expression of OX40L and increased DC expression of TGF-β. This in vitro treatment also resulted in increased Treg TGF-β expression and reduced Th2 responses by CD4⁺ T cells from patients with ABPA. These data provide rationale for a therapeutic trial of vitamin D to prevent or treat ABPA in patients with CF.     

Latent TGF-β–binding protein 4 modifies muscular dystrophy in mice

Most single-gene diseases, including muscular dystrophy, display a nonuniform phenotype. Phenotypic variability arises, in part, due to the presence of genetic modifiers that enhance or suppress the disease process. We employed an unbiased mapping approach to search for genes that modify muscular dystrophy in mice. In a genome-wide scan, we identified a single strong locus on chromosome 7 that influenced two pathological features of muscular dystrophy, muscle membrane permeability and muscle fibrosis. Within this genomic interval, an insertion/deletion polymorphism of 36 bp in the coding region of the latent TGF-β–binding protein 4 gene (Ltbp4) was found. Ltbp4 encodes a latent TGF-β–binding protein that sequesters TGF-β and regulates its availability for binding to the TGF-β receptor. Insertion of 12 amino acids into the proline-rich region of LTBP4 reduced proteolytic cleavage and was associated with reduced TGF-β signaling, decreased fibrosis, and improved muscle pathology in a mouse model of muscular dystrophy. In contrast, a 12-amino-acid deletion in LTBP4 was associated with increased proteolysis, SMAD signaling, and fibrosis. These data identify Ltbp4 as a target gene to regulate TGF-β signaling and modify outcomes in muscular dystrophy.     

Menthol Enhances an Antiproliferative Activity of 1α,25-Dihydroxyvitamin D3 in LNCaP Cells

1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], the most active form of vitamin D₃, and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1α,25(OH)₂D₃ for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1α,25(OH)₂D₃ in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1α,25(OH)₂D₃-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1α,25(OH)₂D₃ does not induce acute Ca²⁺ response, whereas menthol evokes an increase in [Ca²⁺]_i, which suggests that cross-talks of menthol-induced Ca²⁺ signaling with 1α,25(OH)₂D₃-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1α,25(OH)₂D₃ and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1α,25(OH)₂D₃-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1α,25(OH)₂D₃.     

Detection of a new heparin-dependent inhibitor of thrombin in human plasma.

The influence of the serum binding protein (DBP) for vitamin D and its metabolites on the concentration of its main ligands, 25-hydroxyvitamin D₃ (25-OHD₃) and 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂D₃) was studied. The concentration of both 1,25-(OH)₂D₃ and DBP in normal female subjects (45±14 ng/liter and 333±58 mg/liter, mean±SD, respectively; n = 58) increased during the intake of estro-progestogens (69±27 ng/liter and 488±90 mg/liter, respectively; n = 29), whereas the 25-OHD₃ concentration remained unchanged. A positive correlation was found between the concentrations of 1,25-(OH)₂D₃ and DBP in these women.     

Effect of Vitamin D Status on the Equilibrium between Occupied and Unoccupied 1,25-Dihydroxyvitamin D Intestinal Receptors in the Chick

The dynamic equilibrium between in vivo occupied and unoccupied 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] receptors of the chick intestinal mucosa was investigated by the exchange assay previously reported [(1980). J. Biol. Chem. 255: 9534-9537]. These parameters and their correlation to biological response, i.e., the levels of intestinal vitamin D-dependent calcium binding protein (CaBP), were assessed under different physiological conditions. After a single 1,25(OH)₂D₃ injection (3.25 nmol), occupied receptor levels increased sharply to a maximum between 1 and 2 h, followed by a rapid decline. A single dose of 1α-hydroxy-vitamin D₃ [1α(OH)D₃], an analog that requires 25-hydroxylation for biological activity, resulted in a protracted, albeit lower, response with maximal receptor occupancy at 6 h and half maximal levels 24 h after injection. The intestinal receptor occupancy patterns mirrored the serum 1,25(OH)₂D₃ levels after either 1,25(OH)₂D₃ or 1α(OH)D₃ treatment. Additionally, time-course (half-life) of blood disappearance of 1,25(OH)₂D₃ and occupied receptor levels were similar (1.9 and 2.3 h, respectively), suggesting that the amount of occupied 1,25(OH)₂D₃ receptor is determined by a simple equilibrium between serum 1,25(OH)₂D₃ and unoccupied receptors. A dose-response study after intramuscular 1,25(OH)₂D₃ injection yielded a hyperbolic curve with an apparent plateau at 70% receptor occupancy, corresponding to 5 nmol 1,25(OH)₂D₃ injected. Half-maximal occupancy was reached after a dose of 1 nmol 1,25(OH)₂D₃, corresponding to 1.5 ng 1,25(OH)₂D₃/ml serum. From this value the apparent K_d in vivo is 3.7 nM, which is similar to that determined in vitro. A 10-fold increase in the 1α(OH)D₃ dose resulted in less than a doubling of the levels of serum 1,25(OH)₂D₃, occupied 1,25(OH)₂D₃ receptors, or CaBP. Under all experimental conditions, there was a positive correlation between occupied receptor and CaBP levels; however, the slope of the lines depended on the times chosen for the assays due in part to the lag period for CaBP induction and its accumulation within the cell. Conversely, the correlation between serum 1,25-(OH)₂D₃ levels and occupied receptor levels yielded a single regression line independent of the observation time. Short and long-term treatment with different vitamin D metabolites, estrogen, progesterone, or cortisol did not affect the levels of total intestinal 1,25(OH)₂D₃ receptor. Under normal physiological conditions, only 10-15% of the total 1,25(OH)₂D₃ receptor population was occupied by ligand. These studies provide a basis for further investigations of physiological and biochemical parameters of the vitamin D endocrine system and their clinical applications.     

Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells

Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133⁺ TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG–dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2^+/– mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.     

Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-β mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-β receptor (TβRII), which in turn promotes a TβRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TβRII is considered the principal regulatory mechanism of TβRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TβRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TβRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TβRII tail tyrosine residues, resulting in inhibition of TβR-dependent fibrotic signaling. The collagen-binding receptor integrin α1β1 was required for recruitment of TCPTP to the TβRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TβRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1β1 and TβRII that is essential for TβRII-mediated SMAD activation and fibrotic signaling pathways.     

Targeted 25-hydroxyvitamin D3 1α-hydroxylase Adoptive Gene Therapy Ameliorates DSS-induced Colitis Without Causing Hypercalcemia in Mice

Systemic 1,25(OH)₂D₃ treatment ameliorating murine inflammatory bowel diseases (IBD) could not be applied to patients because of hypercalcemia. We tested the hypothesis that increasing 1,25(OH)₂D₃ synthesis locally by targeting delivery of the 1α-hydroxylase gene (CYP27B1) to the inflamed bowel would ameliorate IBD without causing hypercalcemia. Our targeting strategy is the use of CD11b⁺/Gr1⁺ monocytes as the cell vehicle and a macrophage-specific promoter (Mac1) to control CYP27B1 expression. The CD11b⁺/Gr1⁺ monocytes migrated initially to inflamed colon and some healthy tissues in dextran sulfate sodium (DSS) colitis mice; however, only the migration of monocytes to the inflamed colon was sustained. Adoptive transfer of Gr1⁺ monocytes did not cause hepatic injury. Infusion of Mac1-CYP27B1-modified monocytes increased body weight gain, survival, and colon length, and expedited mucosal regeneration. Expression of pathogenic Th17 and Th1 cytokines (interleukin (IL)-17a and interferon (IFN)-α) was decreased, while expression of protective Th2 cytokines (IL-5 and IL-13) was increased, by the treatment. This therapy also enhanced tight junction gene expression in the colon. No hypercalcemia occurred following this therapy. In conclusion, we have for the first time obtained proof-of-principle evidence for a novel monocyte-based adoptive CYP27B1 gene therapy using a mouse IBD model. This strategy could be developed into a novel therapy for IBD and other autoimmune diseases.     

Extraction of Vitamin D Metabolites by Bones of Normal Adult Dogs

Using the isolated perfused canine tibia we examined the extraction of [³H]25(OH)D₃, [³H]1,25(OH)₂D₃ and [³H]24,25(OH)₂D₃ by bone of normal adult dogs. The studies were performed with and without vitamin D binding protein (DBP) in the perfusate to examine the effect of protein binding on the extraction of the vitamin D metabolites. An average of 48±2% of [³H]25(OH)D₃ was extracted by bone, when no DBP was present. However, addition of only a small amount of DBP (~720 ng/ml of perfusate) nearly completely abolished the extraction of [³H]25(OH)D₃ by bone. No degradation and/or transformation of the labeled 25(OH)D₃ could be demonstrated during passage through the isolated perfused bone. The extraction of [³H]24,25(OH)₂D₃ in a DBP-free medium averaged 33±5%. Addition of 720 ng of DBP/ml of perfusate completely inhibited the extraction of this metabolite. The extraction of [³H]1,25(OH)₂D₃ averaged 30±3% in a DBP free medium and no inhibition of the extraction was demonstrated after addition of DBP (720 ng/ml of perfusate). However, addition of DBP in a concentration of 14.4 μg/ml of perfusate reduced the extraction of 1,25(OH)₂D₃ to 8±2%, a value still significantly higher than that seen after addition of 20 times less DBP to perfusions with 25(OH)D₃ and 24,25(OH)₂D₃. It is concluded that the isolated perfused bone of normal dogs can extract significant amounts of 25(OH)D₃, 1,25(OH)₂D₃, and 24,25(OH)₂D₃. Small concentrations of DBP (720 ng/ml) in the perfusate significantly inhibited the extraction of 25(OH)D₃ and 24,25(OH)₂D₃. A carrier role for DBP is suggested and it is proposed that the levels of free vitamin D are important for extraction of the metabolites by bone. Therefore, due to the different affinities of DBP for the various metabolites of vitamin D, only 1,25(OH)₂D₃ is extracted in vitro in significant amounts by bone of normal adult dogs, in the presence of DBP.     

miR-181b targets semaphorin 3A to mediate TGF-β–induced endothelial-mesenchymal transition related to atrial fibrillation

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β–treated human AEECs, cardiac-specific TGF-β–transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β–treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β–transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.     

Retracted Article: TMEM88 inhibits fibrosis in renal proximal tubular epithelial cells by suppressing the transforming growth factor-β1/Smad signaling pathway

Transmembrane protein 88 (TMEM88) belongs to a member of the TMEM family, and was reported to be involved in fibrogenesis. However, the biological role of TMEM88 in renal fibrosis has not been elucidated. Therefore, the objective of this study was to investigate the effect of TMEM88 on cell proliferation and extracellular matrix (ECM) accumulation in a TGF-β1-induced human renal proximal tubular epithelial cell line (HK2). Our results showed that TMEM88 was downregulated in renal fibrotic tissues and TGF-β1-treated HK2 cells. In addition, TMEM88 overexpression inhibited TGF-β1-induced cell proliferation and migration in HK2 cells. Furthermore, TMEM88 overexpression reduced the production of α-SMA, collagen I, and collagen III in TGF-β1-stimulated HK2 cells. Mechanistically, TMEM88 overexpression suppressed the phosphorylation status of Smad2 and Smad3 in TGF-β1-stimulated HK2 cells. In conclusion, data from our experiments demonstrate that TMEM88 plays a pivotal role in the pathological process of renal fibrosis. TMEM88 inhibited fibrosis in renal proximal tubular epithelial cells by suppressing the TGF-β1/Smad signaling pathway.

Transmembrane protein 88 (TMEM88) belongs to a member of the TMEM family, and was reported to be involved in fibrogenesis.     

DLC1-dependent parathyroid hormone–like hormone inhibition suppresses breast cancer bone metastasis

Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β–induced expression of parathyroid hormone–like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho–TGF-β crosstalk in osteolytic bone metastasis.     

Blood pressure homeostasis is maintained by a P311–TGF-β axis

P311 is an 8-kDa intracellular protein that is highly conserved across species and is expressed in the nervous system as well as in vascular and visceral smooth muscle cells. P311-null (P311^–/–) mice display learning and memory defects, but alterations in their vasculature have not been previously described. Here we report that P311^–/– mice are markedly hypotensive with accompanying defects in vascular tone and VSMC contractility. Functional abnormalities in P311^–/– mice resulted from decreased total and active levels of TGF-β1, TGF-β2, and TGF-β3 that arise as a specific consequence of decreased translation. Vascular hypofunctionality was fully rescued in vitro and in vivo by exogenous TGF-β1–TGF-β3. Conversely, P311-transgenic (P311^TG) mice had elevated levels of TGF-β1–TGF-β3 and subsequent hypertension. Consistent with findings attained in mouse models, arteries recovered from hypertensive human patients displayed increased P311 expression. Thus, we identified P311 as the first protein known to modulate TGF-β translation and the first pan-regulator of TGF-β expression under steady-state conditions. Together, our findings point to P311 as a critical blood pressure regulator and establish a potential link between P311 expression and the development of hypertensive disease.