NLRP3 Attenuates Intraocular Inflammation by Inhibiting AIM2-Mediated Pyroptosis Through the Phosphorylated Salt-Inducible Kinase 1/Sterol Regulatory Element Binding Transcription Factor 1 Pathway.

OBJECTIVE: The NLRP3 inflammasome has been shown to be involved in the development of uveitis, but the exact mechanism remains elusive. This study was undertaken to explore the role of NLRP3 in the development of uveitis. METHODS: First, Nlrp3-deficient mice were used to study the role of NLRP3 in experimental autoimmune diseases, such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE). Next, the gathering of ASC, activation of caspase 1 and gasdermin D, and secretion of lactate dehydrogenase and interleukin-1ß were detected to confirm macrophage pyroptosis and AIM2 activation in the Nlrp3-/- mice. Additionally, RNA sequencing and chromatin immunoprecipitation-polymerase chain reaction were used to investigate the phosphorylated salt-inducible kinase 1 (p-SIK1)/sterol regulatory element binding transcription factor 1 (SREBF1) pathway, which regulates the transcription of Aim2. Finally, overexpression of Nlrp3 was applied to treat EAU. RESULTS: Surprisingly, our findings show that NLRP3 plays an antiinflammatory role in 2 models of EAU and EAE. Additionally, macrophages show an increased M1 activation and pyroptosis in Nlrp3-/- mice. Further experiments indicate that this pyroptosis of macrophages was mediated by the up-regulated transcription of Aim2 as a result of Nlrp3 deficiency. In mechanistic studies, Nlrp3 deficiency was implicated in the down-regulation of p-SIK1 and subsequently the up-regulation of SREBF1, which binds to Aim2 and then promotes the latter's transcription. Finally, Aim2 deficiency, RNA silencing of Aim2 or Srebf1, and overexpression of Nlrp3 resulted in attenuated inflammation of EAU. CONCLUSION: Our data demonstrate that NLRP3 inhibits AIM2 inflammasome-mediated EAU by regulating the p-SIK1/SREBF1 pathway, highlighting the therapeutic potential of targeting Nlrp3.

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein.

Factor (F) XII, a key component of the contact system, triggers clotting via the intrinsic pathway, and is implicated in propagating thrombosis. Although nucleic acids are potent activators, it is unclear how the contact system is regulated to prevent uncontrolled clotting. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa and attenuates its capacity to trigger coagulation. To investigate the role of HRG as a regulator of the intrinsic pathway, we compared RNA- and DNA-induced thrombin generation in plasma from HRG-deficient and wild-type mice. Thrombin generation was enhanced in plasma from HRG-deficient mice, and accelerated clotting was restored to normal with HRG reconstitution. Although blood loss after tail tip amputation was similar in HRG-deficient and wild-type mice, carotid artery occlusion after FeCl3 injury was accelerated in HRG-deficient mice, and HRG administration abrogated this effect. To confirm that HRG modulates the contact system, we used DNase, RNase, and antisense oligonucleotides to characterize the FeCl3 model. Whereas DNase or FVII knockdown had no effect, carotid occlusion was abrogated with RNase or FXII knockdown, confirming that FeCl3-induced thrombosis is triggered by RNA in a FXII-dependent fashion. Therefore, in a nucleic acid-driven model, HRG inhibits thrombosis by modulating the intrinsic pathway of coagulation.

Underactivation of the adiponectin-adiponectin receptor 1 axis in clear cell renal cell carcinoma: implications for progression.

Energy-sensing pathways, normally coordinated by 5' AMP-activated protein kinase (AMPK), are dysregulated in renal cell carcinoma (RCC). Obesity can accentuate the pre-existing pro-tumorigenic metabolic machinery in RCC cells through its associated obesogenic hormonal milieu, characterized by lower circulating levels of adiponectin. In RCC patients, low adiponectin levels associate clinically with more aggressive disease. We investigated the adiponectin signaling pathway in RCC, focusing on adiponectin receptor 1 (AdipoR1) and associated activation of AMPK. AdipoR1 protein in RCC and normal surrounding renal tissues was determined by Western blot analysis and immunohistochemistry. Anti-tumorigenic effects of adiponectin in RCC cells in vitro were investigated via VEGF and MMP ELISA and invasion assays. Using in vivo models of RCC, the effect of AdipoR1-knockdown (shRNA) on tumor latency, growth and dissemination were determined. AdipoR1 protein was significantly reduced in clear cell RCC specimens. Adiponectin treatment inhibited VEGF, MMP-2 and MMP-9 secretion and activity and invasive and migratory capacities of RCC cells. AMPKα1-knockdown (shRNA) attenuated adiponectin's effects. In cells stably expressing AdipoR1-specific shRNA, AMPK activation by adiponectin was significantly reduced compared to cells expressing control shRNA. In vivo, AdipoR1 knockdown increased the growth, dissemination and angiogenesis of RCC. These findings suggest that deficiencies in the entire adiponectin hormonal axis (the hormone and its receptor) result in underactivation of AMPK leading to increased angiogenic and invasive capacities of RCC. The established link between obesity and RCC can therefore be further explained by the adiponectin deficiency in obese individuals together with reduced AdipoR1 protein in RCC.

Expression and vitamin D3 regulation of long-chain fatty-acid-CoA ligase 3 in human prostate cancer cells.

We found previously that long-chain fatty-acid-CoA ligase 3 (FACL3), a critical enzyme for activation of long-chain fatty acids, was upregulated by 1α, 25(OH)(2)D(3) at an mRNA and enzyme activity levels in prostate cancer cells. Our further study indicated that the FACL3 mediated 1α,25(OH)(2)D(3) inhibition of fatty acid synthase (FAS), which is associated with many cancers, including prostate cancer. In the current study, we investigated an FACL3 protein expression and its regulation by 1α, 25(OH)(2)D(3) and its synthetic analogs EB1089 and CB1093 in prostate cancer cells. The results showed that the expression of an FACL3 protein was upregulated by 1α, 25(OH)(2)D(3), EB1089 and CB1093 in LNCaP cells, consistent with their upregulation of an FACL3 mRNA expression. In addition, the FACL3 expression was found to be markedly low at both mRNA and protein levels in more transformed prostate cancer PC-3 and DU145 cells compared with less transformed LNCaP cells. The data suggest that decreased FACL3 expression might be associated with a more malignant phenotype of prostate cancer.

Inter-related in vitro effects of androgens, fatty acids and oxidative stress in prostate cancer: a mechanistic model supporting prevention strategies.

Oxidation of mitochondrial fatty acids (FA) results in the generation of reactive oxygen species (ROS) which have been postulated to play a key role in the initiation and progression of prostate cancer (PC). We previously reported that androgens increase FA uptake into PC cells. We thus examined if androgens that are known to induce ROS generation regulate FA oxidation in PC cells. The effects of the androgen-depleted medium, R1881 (synthetic androgen) and/or androgen receptor blocker, bicalutamide were examined in the human androgen-responsive but not dependent 22rv1 cells. R1881 supplementation significantly increased mitochondrial FA oxidation ((14)C-radiolabeled FA degradation studies), resulting in increased ROS production. Androgens increased the mRNA levels of carnitine palmitoyltransferase (CPT1), the rate limiting enzyme in the process of mitochondrial FA oxidation. Treatment with R1881 and bicalutamide inhibited these androgen regulated effects. Inhibition of mitochondrial ROS generation by two different inhibitors, rotenone and thenoyltrifluoroacetone, eliminated the androgen-induced ROS generation, to the same level as in cells deprived of androgens or treated with R1881 and bicalutamide. Taken together, androgens increase the mitochondrial oxidation of FA, leading to increased production of ROS that is associated with prostate cell proliferation and mutagenesis. These results therefore support the rationale for PC prevention using 5-alpha reductase inhibitors, dietary restrictions or anti-oxidants, each of which has different inhibitory but complementary effects.

Thymus-derived glucocorticoids mediate androgen effects on thymocyte homeostasis.

Androgens contribute to the involution process of the aging thymus gland. However, molecular mechanisms behind this effect remain largely unknown. We have investigated the influence of testosterone on the ectopic synthesis of glucocorticoids (GCs) in thymocytes, an activity recently shown by us to be important for the homeostatic regulation of these cells. Castration, which leads to a strong increase in thymus tissue and function, was associated with a reduced GC release from thymocytes caused by down-regulated expression of several enzymes involved in GC synthesis, without affecting GC synthesis in the adrenals. Testosterone treatment of castrated male mice reversed these effects, also without affecting adrenal GC synthesis. The effects of testosterone in castrated mice on thymocyte homeostasis and GC release were strongly reduced in mice pretreated with the CYP11B1 enzyme inhibitor metyrapone, acting on the last step in the corticosterone synthesis. The androgen-induced thymic involution was dependent on GC action, because this was completely absent in mice lacking GC receptor (GR) expression specifically in thymocytes. We provide here an unrecognized mechanism how androgens contribute to thymic involution by stimulating local synthesis and release of GCs in the thymus.

25-Hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand.

25-Hydroxyvitamin D(3) 1alpha-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), a vitamin D receptor ligand. 25-Hydroxyvitamin D(3) has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1alpha-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D(3) has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D(3) internalisation, in Cyp27b1(-/-) cells explains their higher sensitivity to 25-hydroxyvitamin D(3). 25-Hydroxyvitamin D(3) action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D(3). Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D(3) in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D(3) with 1alpha,25-dihydroxyvitamin D(3) in Cyp27b1(-/-) cells further demonstrates the agonistic action of 25-hydroxyvitamin D(3) and suggests that a synergism between 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) might be physiologically important. In conclusion, 25-hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.

Thymocyte-synthesized glucocorticoids play a role in thymocyte homeostasis and are down-regulated by adrenocorticotropic hormone.

Thymocytes from adult mice synthesize glucocorticoids (GCs), and some data indicate a role for this hormone production in thymic homeostasis. Here we present further support for this view by showing that the dramatic increase in thymocyte number seen after adrenalectomy (ADX) does not correlate with the decrease in systemic GCs but rather with an ACTH-mediated down-regulation of GC synthesis in thymocytes. High ACTH concentrations caused by ADX in wild-type mice down-regulated CYP11B1 mRNA expression, encoding the last enzyme required for corticosterone synthesis and as a consequence reduced GC synthesis in thymocytes. This was not seen in IL-1beta/IL-18 double-knockout mice unable to respond to ADX with high ACTH levels. However, if ADX IL-1beta/IL-18 double-knockout mice were treated with ACTH, this led to a down-regulation of CYP11B1 and GC synthesis in thymocytes. In addition, in vivo treatment of mice with the CYP11B1 antagonist metyrapone, without affecting the systemic corticosterone level, increased thymocyte numbers and in vitro treatment of isolated thymocytes prevented thymocyte loss. Furthermore, in vitro experiments showed that both ACTH and its receptor-induced second-messenger molecule cAMP down-regulated mRNA expression of critical enzymes in GC steroidogenesis and GC synthesis in thymocytes. We conclude that thymocyte-produced GCs are important for the homeostasis of adult mouse thymocytes and that high ACTH level, in contrast to stimulating GC synthesis in the adrenal glands, has the opposite effect in thymocytes.

Age-related synthesis of glucocorticoids in thymocytes.

Glucocorticoids (GCs) are primarily synthesized in the adrenal glands but an ectopic production has also been reported in the brain, the gastrointestinal tract and in thymic epithelial cells (TEC). Here we show that thymocytes express genes encoding for all enzymes required for de novo GC synthesis and produce the hormone as demonstrated by both a GC specific reporter assay and a corticosterone specific ELISA assay. Interestingly, GC synthesis is detectable in cells from young mice (4 weeks) and thereafter increases during aging (14-22 weeks) together with an increased gene expression of the rate-limiting enzymes StAR and CYP11A1. Hormone production occurred at a thymocyte differentiation stage characterized by being double positive for the CD4 and CD8 surface markers but was found to be unrelated to CD69 expression, a marker for thymocytes undergoing positive selection. No GC synthesis was found in resting or anti-CD3 activated CD4 and CD8 positive T cells isolated from the spleen. Thymocyte-derived GC had an anti-proliferative effect on a GR-transfected cell line and induced apoptosis in thymocytes. The age- and differentiation stage-related GC synthesis in thymocytes may play a role in the involution process that the thymus gland undergoes.

Vitamin D3 inhibits fatty acid synthase expression by stimulating the expression of long-chain fatty-acid-CoA ligase 3 in prostate cancer cells.

FAS and FACL3 are enzymes of fatty acid metabolism. In our previous studies, we found that FAS and FACL3 genes were vitamin D3-regulated and involved in the antiproliferative effect of 1alpha,25(OH)2D3 in the human prostate cancer LNCaP cells. Here, we elucidated the mechanism behind the downregulation of FAS expression by vitamin D3. Triacsin C, an inhibitor of FACL3 activity, completely abolished the downregulation of FAS expression by vitamin D3, whereas an inhibitor of FAS activity, cerulenin, had no significant effect on the upregulation of FACL3 expression by vitamin D3 in LNCaP cells. In human prostate cancer PC3 cells, in which FACL3 expression is not regulated by vitamin D3, no regulation of FAS expression was seen. This suggests that the downregulation of FAS expression by vitamin D3 is mediated by vitamin D3 upregulation of FACL3 expression. Myristic acid, one of the substrates preferential for FACL3, enhanced the repression of FAS expression by vitamin D3. The action of myristic acid was abrogated by inhibition of FACL3 activity, suggesting that the enhancement in the downregulation of FAS expression by vitamin D3 is due to the formation of myristoyl-CoA. The data suggest that vitamin D3-repression of FAS mRNA expression is the consequence of feedback inhibition of FAS expression by long chain fatty acyl-CoAs, which are formed by FACL3 during its upregulation by vitamin D3 in human prostate cancer LNCaP cells.

Calcitriol-induced prostate-derived factor: autocrine control of prostate cancer cell growth.

Calcitriol (1alpha,25-dihydroxycholecalciferol) seems to play an important role in the complex control of prostate cell growth. It inhibits proliferation and induces differentiation and apoptosis in prostate cancer cells. However, the molecular mechanisms of the antiproliferative activity of calcitriol are not completely understood. The expression of prostate-derived factor (PDF), a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be associated with proapoptotic and antimitotic activities. We show that calcitriol induces PDF expression in LNCaP human prostate cancer cells in a concentration- and time-dependent manner. In LNCaP cells, the suppression of cell growth by calcitriol is accompanied by stimulation of PDF mRNA and protein synthesis. Human recombinant PDF inhibits LNCaP cell growth. We do not detect any effect of PDF-specific antibody on the basal growth of LNCaP cells, but this antibody partially reverses the suppression of LNCaP cell growth by calcitriol, suggesting that the effect of calcitriol on cell growth is at least partially mediated by PDF. In PC-3 cells, which are less responsive to the growth-inhibitory effect of calcitriol, it has no effect on PDF expression. We do not detect an effect of recombinant PDF on SMAD phosphorylation in LNCaP cells, but ERK1/2 kinases are transiently phosphorylated in response to PDF, which suggests that in LNCaP cells PDF may exert its action through pathway alternative to the classical TGF-beta signaling pathway. The present study describes the regulation of PDF, the proapoptotic protein of the TGF-beta superfamily, by calcitriol in androgen-responsive prostate cancer cells. This is a new link between calcitriol and growth factors of TGF-beta superfamily in the control of prostate cell growth.

The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth.

The antiproliferative effect of 1alpha,25(OH)(2)D(3) on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.

Inhibition of fatty acid synthase expression by 1alpha,25-dihydroxyvitamin D3 in prostate cancer cells.

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) and its derivatives are a potential treatment of human prostate cancer. The antiproliferative action of 1alpha,25(OH)(2)D(3) is mainly exerted through nuclear vitamin D receptor (VDR)-mediated control of target gene transcription. To explore the target genes which are regulated by 1alpha,25(OH)(2)D(3) in human prostate cancer LNCaP cells, cDNA microarray was performed by using a chip that contains 3000 gene probes. The results showed that 24 genes were regulated by 1alpha,25(OH)(2)D(3). Five of them encode proteins which belong to metabolic enzymes and fatty acid biosynthesis. Fatty acid synthase (FAS) was found to be down-regulated by 1alpha,25(OH)(2)D(3), and the regulation was confirmed by real-time quantitative RT-PCR analysis. Inhibition of FAS expression by 1alpha,25(OH)(2)D(3) in LNCaP cells was more than 50% at 6h. Inhibitory effect of 1alpha,25(OH)(2)D(3) on FAS expression was completely blocked in the presence of protein synthesis inhibitor cycloheximide, indicating that the down-regulation of FAS gene expression by 1alpha,25(OH)(2)D(3) was indirect in LNCaP cells. An inhibition of FAS activity by cerulenin resulted in a strong inhibition of LNCaP cell proliferation. The inhibition of FAS expression and cell proliferation by 1alpha,25(OH)(2)D(3) seemed to be androgen-dependent, since antiandrogen, casodex and DCC-treatment of serum blocked the vitamin D action. The findings suggest that FAS is involved in the antiproliferative effect of 1alpha,25(OH)(2)D(3) in presence of androgens on prostate cancer LNCaP cells.