Bryan S. Benn

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k.

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

Vitamin D: molecular mechanism of action.

Abstract:  Vitamin D maintains calcium homeostasis and is required for bone development and maintenance. Recent evidence has indicated an interrelationship between vitamin D and health beyond bone, including effects on cell proliferation and on the immune system. New developments in our lab related to the function and regulation of target proteins have provided novel insights into the mechanisms of vitamin D action. Studies in our lab have shown that the calcium‐binding protein, calbindin, which has been reported to be a facilitator of calcium diffusion, also has an important role in protecting against apoptotic cell death in different tissues including protection against cytokine destruction of osteoblastic and pancreatic β cells. These findings have important implications for the therapeutic intervention of many disorders including diabetes and osteoporosis. Recent studies in our laboratory of intestinal calcium absorption using calbindin‐D9k null mutant mice as well as mice lacking the 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) inducible epithelial calcium channel, TRPV6, provide evidence for the first time of calbindin‐D9k and TRPV6 independent regulation of active calcium absorption. Besides calbindin, the other major target of 1,25(OH)2D3 in intestine and kidney is 25(OH)D3 24 hydroxylase (24(OH)ase), which is involved in the catabolism of 1,25(OH)2D3. In our laboratory we have identified various factors that cooperate with the vitamin D receptor in regulating 24(OH)ase expression including C/EBP β, SWI/SNF (complexes that remodel chromatin using the energy of ATP hydrolysis) and the methyltransferases, CARM1 and G9a. Evidence is also presented for C/EBP β as a nuclear coupling factor that coordinates regulation of osteopontin by 1,25(OH)2D3 and PTH. Our findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)2D3 mediates its multiple biological effects.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

IFN-γ–Producing T-Helper 17.1 Cells Are Increased in Sarcoidosis and Are More Prevalent than T-Helper Type 1 Cells

RATIONALE Pulmonary sarcoidosis is classically defined by T-helper (Th) cell type 1 inflammation (e.g., IFN-γ production by CD4(+) effector T cells). Recently, IL-17A-secreting cells have been found in lung lavage, invoking Th17 immunity in sarcoidosis. Studies also identified IL-17A-secreting cells that expressed IFN-γ, but their abundance as a percentage of total CD4(+) cells was either low or undetermined. OBJECTIVES Based on evidence that Th17 cells can be polarized to Th17.1 cells to produce only IFN-γ, our goal was to determine whether Th17.1 cells are a prominent source of IFN-γ in sarcoidosis. METHODS We developed a single-cell approach to define and isolate major Th-cell subsets using combinations of chemokine receptors and fluorescence-activated cell sorting. We subsequently confirmed the accuracy of subset enrichment by measuring cytokine production. MEASUREMENTS AND MAIN RESULTS Discrimination between Th17 and Th17.1 cells revealed very high percentages of Th17.1 cells in lung lavage in sarcoidosis compared with controls in two separate cohorts. No differences in Th17 or Th1 lavage cells were found compared with controls. Lung lavage Th17.1-cell percentages were also higher than Th1-cell percentages, and approximately 60% of Th17.1-enriched cells produced only IFN-γ. CONCLUSIONS Combined use of surface markers and functional assays to study CD4(+) T cells in sarcoidosis revealed a marked expansion of Th17.1 cells that only produce IFN-γ. These results suggest that Th17.1 cells could be misclassified as Th1 cells and may be the predominant producer of IFN-γ in pulmonary sarcoidosis, challenging the Th1 paradigm of pathogenesis.

Mechanisms Involved in Vitamin D Mediated Intestinal Calcium Absorption and in Non-Classical Actions of Vitamin D

Recent studies in our laboratory using calbindin-D9k null mutant mice as well as mice lacking the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inducible epithelial calcium channel TRPV6 provide evidence for calbindin-D9k and TRPV6 independent regulation of active intestinal calcium absorption. These findings suggest that in the knock out (KO) mice there is compensation by another calcium channel or protein and that other novel factors are involved in 1,25(OH)2D3 mediated active intestinal calcium absorption. In addition, 1,25(OH)2D3 mediated paracellular transport of calcium may have contributed to the normalization of serum calcium in the null mutant mice. 1,25(OH)2D3 downregulates cadherin-17 and upregulates claudin-2 and claudin-12 in the intestine, suggesting that 1,25(OH)2D3, by regulating these epithelial cell junction proteins, can route calcium through the paracellular path. With regard to non-classical actions, 1,25(OH)2D3 has been reported to inhibit the proliferation of a number of malignant cells and to regulate adaptive as well as innate immunity. This article will review new developments related to the function and regulation of vitamin D target proteins in classical and non-classical vitamin D target tissues that have provided novel insight into mechanisms of vitamin D action.

Phospholipase C-Mediated Regulation of Transient Receptor Potential Vanilloid 6 Channels: Implications in Active Intestinal Ca2+ Transport

Transient receptor potential vanilloid 6 (TRPV6) channels play an important role in intestinal Ca2+ transport. These channels undergo Ca2+-induced inactivation. Here we show that Ca2+ flowing through these channels activates phospholipase C (PLC) leading to the depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) and formation of inositol 1,4,5-trisphosphate in TRPV6-expressing cells. PIP2 depletion was inhibited by the two structurally different PLC inhibitors 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and edelfosine. Ca2+-induced inactivation of TRPV6 was also prevented by the PLC inhibitors in whole-cell patch-clamp experiments. Ca2+ signals in TRPV6-expressing cells were transient upon restoration of extracellular Ca2+ but were rendered more sustained by the PLC inhibitors. Finally, intestinal Ca2+ transport in the everted duodenal sac assay was enhanced by edelfosine. These observations suggest that Ca2+-induced inactivation of TRPV6 limits intestinal Ca2+ absorption and raise the possibility that Ca2+ absorption can be enhanced pharmacologically by interfering with PLC activation.

New Insights into the Function and Regulation of Vitamin D Target Proteins

Calbindin-D(28k) has been reported to be a facilitator of calcium diffusion and to protect against apoptotic cell death. Most recently, we found that the presence of calbindin-D(28k) results in reduced calcium influx through voltage-dependent L-type Ca(2+) channels and enhanced sensitivity of the channels to calcium dependent inactivation. Co-immunoprecipitation and GST pull down assays indicate that calbindin-D(28k) interacts with the C-terminus of the L-type calcium channel alpha(1c) subunit (Ca(v)1.2). This is the first report of the binding of calbindin to a calcium channel and provides new insight concerning mechanisms by which calbindin acts to modulate intracellular calcium. Besides calbindin, another major target of 1,25(OH)(2)D(3) is 24(OH)ase, which is involved in the catabolism of 1,25(OH)(2)D(3). We reported that C/EBPbeta is a major transcriptional activator of 24(OH)ase that cooperates with CBP/p300 in regulating VDR mediated 24(OH)ase transcription. Recently, we found, in addition to p160 coactivators, that SWI/SNF complexes (that facilitate transcription by remodeling chromatin using the energy of ATP hydrolysis) are also involved in VDR mediated 24(OH)ase transcription and functionally cooperate with C/EBPbeta in regulating 24(OH)ase. These findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)(2)D(3) mediates its multiple biological effects.

New Insights into the Mechanisms Involved in the Pleiotropic Actions of 1,25Dihydroxyvitamin D3

Abstract:  Vitamin D functions to regulate calcium homeostasis in intestine, kidney, and bone. Vitamin D deficiency during bone development causes rickets and in adults vitamin D deficiency, which has been shown to be common in the elderly population, can cause secondary hyperparathyroidism that can result in osteomalacia and increased risk of fracture. Recent evidence has suggested that vitamin D can have numerous other physiological functions including protection against certain autoimmune diseases, such as diabetes and multiple sclerosis and inhibition of proliferation of a number of malignant cells including breast and prostate cancer cells. Exactly how vitamin D affects numerous different systems is a subject of continuing investigation. This article will review new developments related to the function and regulation of vitamin D target proteins in classic vitamin D target tissues that have provided novel insight into the mechanism of vitamin D action.

Mechanism of Action of 1,25-Dihydroxyvitamin D3 on Intestinal Calcium Absorption and Renal Calcium Transport

In the intestine 1,25-dihydroxyvitamin D3 [1,25(OH)2D] induces various aspects of the transcellular active calcium transport system including calbindin, the basolateral plasma membrane pump, and the epithelial calcium channel, TRPV6. Calcium can enter the enterocyte through the epithelial calcium channel and then bind to calbindin and move through the cytosol. At the basolateral membrane calcium is transported actively by the plasma membrane calcium pump into the extracellular space. There is increasing evidence that, in the intestine, 1,25(OH)2D can also enhance paracellular calcium diffusion.

Nivolumab-Induced Granulomatous Inflammation of the Pleura

To the Editor: It is with great enthusiasm that we present the first reported case of nivolumab-induced granulomatous inflammation of the pleura diagnosed by medical thoracoscopy. A 61-year-old man with a history of myasthenia gravis and metastatic melanoma to his lungs diagnosed on June 3, 2015, was evaluated in our interventional pulmonology clinic for a recurrent pleural effusion. His history was pertinent for treatment with nivolumab, with the first infusion occurring on August 11, 2015, and dosed as 2 mg/kg actual body weight, with subsequent infusions on October 1, October 15, and October 29. In May 2016, the patient presented with worsening dyspnea, with a restaging positron emission tomography–computed tomography scan on May 5, 2016, showing new bilateral pleural effusions. Right-sided ultrasound-guided thoracentesis was performed on June 17, 2016; it revealed an exudative effusion with 45% lymphocytes without evidence of malignancy. Because serial imaging showed persistence of the left-sided pleural effusion, the patient was referred for