Excess Nitric Oxide Activates TRPV1-Ca2+-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor α

Excess nitric oxide (NO) deregulates cholesterol metabolism in macrophage foam cells, yet the underlying molecular mechanism is incompletely understood. To investigate the mechanism, we found that in macrophages, treatment with NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) or diethylenetriamine/nitric oxide induced LXRα degradation and reduced the expression of the downstream target of LXRα, ATP-binding cassette transporter A1 (ABCA1), and cholesterol efflux. In addition, SNAP induced calcium (Ca²⁺) influx into cells, increased calpain activity and promoted the formation of calpain-LXRα complex. Pharmacological inhibition of calpain activity reversed the SNAP-induced degradation of LXRα, down-regulation of ABCA1 and impairment of cholesterol efflux in macrophages. SNAP increased the formation of calpain-LXRα complex in a Pro-Glu-Ser-Thr (PEST) motif-dependent manner. Truncation of the PEST motif in LXRα abolished the calpain-dependent proteolysis. Removal of extracellular Ca²⁺ by EGTA or pharmacological inhibition of TRPV1 channel activity diminished SNAP-induced increase in intracellular Ca²⁺, calpain activation, LXRα degradation, ABCA1 down-regulation and impaired cholesterol efflux. In conclusion, excess NO may activate calpain via TRPV1-Ca²⁺ signaling and promote the recognition of calpain in the PEST motif of LXRα, thereby leading to degradation of LXRα and, ultimately, downregulated ABCA1 expression and impaired ABCA1-dependent cholesterol efflux in macrophages.     

The miR-302c/transforming growth factor-β receptor type-2 axis modulates interleukin-1β-induced degenerative changes in osteoarthritic chondrocytes

Chondrocyte production of catabolic and inflammatory mediators participating in extracellular matrix degradation has been regarded as a central event in osteoarthritis (OA) development. During OA pathogenesis, interleukin-1β (IL-1β) decreases the mRNA expression and protein levels of transforming growth factor-β receptor type-2 (TGFBR2), thus disrupting transforming growth factor-β signaling and promoting OA development. In the present study, we attempted to identify the differentially expressed genes in OA chondrocytes upon IL-1β treatment, investigate their specific roles in OA development, and reveal the underlying mechanism. As shown by online data analysis and experimental results, TGFBR2 expression was significantly downregulated in IL-1β-treated human primary OA chondrocytes. IL-1β treatment induced degenerative changes in OA chondrocytes, as manifested by increased matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 proteins, decreased Aggrecan and Collagen II proteins, and suppressed OA chondrocyte proliferation. These degenerative changes were significantly reversed by TGFBR2 overexpression. miR-302c expression was markedly induced by IL-1β treatment in OA chondrocytes. miR-302c suppressed the expression of TGFBR2 via direct binding to its 3′- untranslated region. Similar to TGFBR2 overexpression, miR-302c inhibition significantly improved IL-1β-induced degenerative changes in OA chondrocytes. Conversely, TGFBR2 silencing enhanced IL-1β-induced degenerative changes and significantly reversed the effects of miR-302c inhibition in response to IL-1β treatment. In conclusion, the miR-302c/TGFBR2 axis could modulate IL-1β-induced degenerative changes in OA chondrocytes and might become a novel target for OA treatment.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00591-2) contains supplementary material, which is available to authorized users.     

SREBP-1c/MicroRNA 33b Genomic Loci Control Adipocyte Differentiation

White adipose tissue (WAT) is essential for maintaining metabolic function, especially during obesity. The intronic microRNAs miR-33a and miR-33b, located within the genes encoding sterol regulatory element-binding protein 2 (SREBP-2) and SREBP-1, respectively, are transcribed in concert with their host genes and function alongside them to regulate cholesterol, fatty acid, and glucose metabolism. SREBP-1 is highly expressed in mature WAT and plays a critical role in promoting in vitro adipocyte differentiation. It is unknown whether miR-33b is induced during or involved in adipogenesis. This is in part due to loss of miR-33b in rodents, precluding in vivo assessment of the impact of miR-33b using standard mouse models. This work demonstrates that miR-33b is highly induced upon differentiation of human preadipocytes, along with SREBP-1. We further report that miR-33b is an important regulator of adipogenesis, as inhibition of miR-33b enhanced lipid droplet accumulation. Conversely, overexpression of miR-33b impaired preadipocyte proliferation and reduced lipid droplet formation and the induction of peroxisome proliferator-activated receptor γ (PPARγ) target genes during differentiation. These effects may be mediated by targeting of HMGA2, cyclin-dependent kinase 6 (CDK6), and other predicted miR-33b targets. Together, these findings demonstrate a novel role of miR-33b in the regulation of adipocyte differentiation, with important implications for the development of obesity and metabolic disease.     

Effects of NR1H3 Genetic Variation on the Expression of Liver X Receptor α and the Progression of Alzheimer's Disease

Alzheimer''s disease (AD) has been postulated to involve defects in the clearance of amyloid-β (Aβ). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aβ. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aβ42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aβ42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aβ42.     

Transforming Growth Factor β1 (TGF-β1) Enhances Expression of Profibrotic Genes through a Novel Signaling Cascade and MicroRNAs in Renal Mesangial Cells

Increased expression of transforming growth factor-β1 (TGF-β1) in glomerular mesangial cells (MC) augments extracellular matrix accumulation and hypertrophy during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. MicroRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF-β1 to enhance the expression of profibrotic genes like collagen. In this study, we found a significant decrease in the expression of miR-130b in mouse MC treated with TGF-β1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-β receptor 1 (TGF-βR1) was identified as a target of miR-130b. Interestingly, the RIK promoter contains three NF-Y binding sites and was regulated by NF-YC. Furthermore, NF-YC expression was inhibited by TGF-β1, suggesting that a signaling cascade, involving TGF-β1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-βR1 to augment expression of TGF-β1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-βR1, as well as the profibrotic genes collagen type IV α 1 (Col4a1), Col12a1, CTGF, and PAI-1 were up-regulated not only in mouse MC treated with TGF-β1 but also in the glomeruli of streptozotocin-injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA- and host gene-mediated amplifying cascade initiated by TGF-β1 that results in the up-regulation of profibrotic factors, such as TGF-βR1 and collagens associated with the progression of DN.     

FOG2 Protein Down-regulation by Transforming Growth Factor-β1-induced MicroRNA-200b/c Leads to Akt Kinase Activation and Glomerular Mesangial Hypertrophy Related to Diabetic Nephropathy

Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-β1 (TGF-β) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF-β are not fully understood. Recently, miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here, we show that TGF-β activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c, both of which target FOG2, an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-β-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-β-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely, miR-200b/c inhibitors attenuated TGF-β-induced decrease in FOG2 expression. Furthermore, miR-200b/c mimics increased the protein content/cell ratio, whereas miR-200b/c inhibitors abrogated the TGF-β-induced increase in protein content/cell. In addition, down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK, which was also through PI3K activation. These data suggest a new mechanism for TGF-β-induced Akt activation through FOG2 down-regulation by miR-200b/c, which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.     

MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

Background

Even though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488.

Results

Human articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1β was also suppressed whereas exposure of TGF-β3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation.

Conclusions

miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.     

MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes

Introduction

Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.

Methods

MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).

Results

In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3''UTR abrogated the suppressive effect of miR125.

Conclusions

Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.     

Continuous Cyclic Mechanical Tension Increases Ank Expression in Endplate Chondrocytes Through the TGF-β1 and p38 Pathway

The normal ANK protein has a strong influence on anti-calcification. It is known that TGF-β1 is also able to induce extracellular inorganic pyrophosphate (ePPi) elaboration via the TGF-β1-induced ank gene expression and the mitogen-activated protein kinase (MAPK) signaling acts as a downstream effector of TGF-β1. We hypothesized that the expression of the ank gene is regulated by mechanics through TGF-β1-p38 pathway. In this study, we investigated the mechanism of short-time mechanical tension-induced ank gene expression. We found that the continuous cyclic mechanical tension (CCMT) increased the ank gene expression in the endplate chondrocytes, and there was an increase in the TGF-β1 expression after CCMT stimulation. The ank gene expression significantly increased when treated by TGF-β1 in a dose-dependent manner and decreased when treated by SB431542 (ALK inhibitor) in a dose-dependent manner. Our study results indicate that CCMT-induced ank gene expressions may be regulated by TGF-β1 and p38 MAPK pathway.Key words: Continuous cyclic mechanical tension (CCMT), Endplate chondrocytes, ank, TGF-β1, p38     

Investigating Conversion of Endplate Chondrocytes Induced by Intermittent Cyclic Mechanical Unconfined Compression in Three-Dimensional Cultures

Mechanical stimulation is known to regulate the calcification of endplate chondrocytes. The Ank protein has a strong influence on anti-calcification by transports intracellular inorganic pyrophosphate (PPi) to the extracellular matrix. It is known that TGF-β1 is able to induce Ank gene expression and protect chondrocyte calcification. Intermittent cyclic mechanical tension (ICMT) could induce calcification of endplate chondrocytes by decrease the expression of Ank gene. In this study, we investigated the relation of intermittent cyclic mechanical unconfined compression (ICMC) and Ank gene expression. We found that ICMC decreased the Ank gene expression in the endplate chondrocytes, and there was an decreased in the TGF-β1 expression after ICMC stimulation. The Ank gene expression significantly increased when treated by transforming growth factor alpha 1 (TGF-β1) in a dose-dependent manner and decreased when treated by SB431542 (ALK inhibitor) in a dose-dependent manner. Our results implicate that ICMC-induced downregulation of Ank gene expression may be regulated by TGF-β1 in end-plate chondrocytes.Key words: Endplate chondrocytes, ICMC, Ank, TGF-β     

miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4

Introduction

miR-146a is one of the first identified miRNAs expressed differentially in osteoarthritis (OA) cartilage. However, the role it plays in OA pathogenesis is not clear. The aim of this study is to identify a molecular target of miR-146a, thereby elucidating its function in chondrocytes during OA pathogenesis.

Methods

Primary chondrocytes from Sprague-Dawley rats were treated with IL-1β before the expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by real-time PCR and/or western blotting. The effect of miR-146a on cellular response to transforming growth factor (TGF)-β1 was quantified by a luciferase reporter harboring TGF-β1 responsive elements and by extracellular signal-regulated kinase assay. The effect of miR-146a on apoptosis was quantified by the TUNEL assay. OA pathogenesis was surgically induced with joint instability in rats, evaluated by histopathological analysis with safranin O staining, and the expression levels of miR-146a, Smad4, and VEGF were quantified using real-time PCR and/or immunohistochemistry.

Results

IL-1β treatment of chondrocytes increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in a time-dependent manner. miR-146a upregulated VEGF expression and downregulated Smad4 expression in chondrocytes, while a miR-146a inhibitor acted in a converse manner. Smad4, a common mediator of the TGF-β pathway, is identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3''-UTR region of its mRNA. Mutation of the binding sequence significantly relieved the inhibition of the Smad4 reporter activity by miR-146a. Furthermore, miR-146a upregulation of VEGF is mediated by Smad4. Expression of miR-146a led to a reduction of cellular responsiveness to TGF-β and an increase of apoptosis rate in chondrocytes. In vivo, cartilage from surgically induced OA rats displayed higher levels of miR-146a and VEGF compared with the sham group. In contrast, Smad4 expression level was lower in the OA group than the sham group.

Conclusion

IL-1β responsive miR-146a is overexpressed in an experimentally induced OA model, accompanied by upregulation of VEGF and downregulation of Smad4 in vivo. miR-146a may contribute to OA pathogenesis by increasing VEGF levels and by impairing the TGF-β signaling pathway through targeted inhibition of Smad4 in cartilage.     

Endothelial Lineage Differentiation from Induced Pluripotent Stem Cells Is Regulated by MicroRNA-21 and Transforming Growth Factor β2 (TGF-β2) Pathways

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.     

miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity

Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor β1 (TGF-β1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-β1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-β1–induced α-SMA expression. These data demonstrate that TGF-β1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-β1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.