Blockade of HMGB1 Reduces Inflammation and Pruritus in Atopic Dermatitis by Inhibiting Skin Fibroblasts Activation.

INTRODUCTION: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by relapsed eczema and serious pruritus. High-mobility group box 1 protein (HMGB1) is a nuclear-binding protein and serves as an alarmin to promote inflammatory responses. METHODS: In this study, we established an AD mouse model by topical use of MC903 on ears and then used a specific HMGB1-binding peptide cIY8 and a HMGB1 inhibitor of glycyrrhizin to investigate HMGB1 on fibroblast activation in the pathogenesis of AD-like symptoms. RESULTS: Topical use of cIY8 and oral use of glycyrrhizin significantly improved the MC903-induced AD-like symptoms and pathological changes of the ears and scratching behavior in an AD mouse model; cIY8 treatment inhibited the higher mRNAs of IL-1α, IL-4, IL-5, IL-13, and IL-31 in the ears. In human fibroblasts, HMGB1 caused nuclear translocation of NF-kB, and the nuclear translocation could be inhibited by pre-treatment of HMGB1 with cIY8, suggesting that NF-κB signaling pathway participates in the HMGB1-induced inflammation of AD in fibroblasts and that cIY8 effectively impedes the function of HMGB1. Glycyrrhizin inhibited the Ca2+ signaling induced by ionomycin in mouse primary fibroblasts. The fibroblast-related proteins of α-SMA, Hsp47, and vimentin and the pruritus-related proteins of IL-33 and periostin were increased in the ears of the AD mouse model, the ratio of EdU incorporation became higher in mouse fibroblasts treated with MC903, and the higher proliferation and inflammatory responses of the fibroblasts could be reversed by glycyrrhizin treatment. CONCLUSIONS: Fibroblast activation by HMGB1 is one of the critical processes in the development of inflammation and pruritus in the AD mouse model. The specific HMGB1-binding peptide cIY8 and the HMGB1 inhibitor glycyrrhizin inactivate skin fibroblasts to alleviate the inflammation and pruritus in the AD mouse model. Peptide cIY8 may be topically used to treat AD patients in the future.

Maternal high-fat diet regulates offspring hepatic ABCG5 expression and cholesterol metabolism via the gut microbiota and its derived butyrate.

Maternal high-fat diet intake has profound effects on the long-term health of offspring, predisposing them to a higher susceptibility to obesity and metabolic dysfunction-associated steatotic liver disease. However, the detailed mechanisms underlying the role of a maternal high-fat diet in hepatic lipid accumulation in offspring, especially at the weaning age, remain largely unclear. In this study, female C57BL/6J mice were randomly assigned to either a high-fat diet or a control diet, and lipid metabolism parameters were assessed in male offspring at weaning. Gut microbiota analysis and targeted metabolomics of short-chain fatty acids (SCFAs) in these offspring were further performed. Both in vivo and in vitro studies were conducted to explore the role of butyrate in hepatic cholesterol excretion in the liver and HepG2 cells. Our results showed that maternal high-fat feeding led to obesity and dyslipidemia, and exacerbated hepatic lipid accumulation in the livers of offspring at weaning. We observed significant decreases in the abundance of the Firmicutes phylum and the Allobaculum genus, known as producers of SCFAs, particularly butyrate, in the offspring of dams fed a high-fat diet. Additionally, maternal high-fat diet feeding markedly decreased serum butyrate levels and down-regulated ATP-binding cassette transporters G5 (ABCG5) in the liver, accompanied by decreased phosphorylated AMP-activated protein kinase (AMPK) and histone deacetylase 5 (HADC5) expressions. Subsequent in vitro studies revealed that butyrate could induce ABCG5 activation and alleviate lipid accumulation via the AMPK-pHDAC5 pathway in HepG2 cells. Moreover, knockdown of HDAC5 up-regulated ABCG5 expression and promoted cholesterol excretion in HepG2 cells. In conclusion, our study provides novel insights into how maternal high-fat diet feeding inhibits hepatic cholesterol excretion and down-regulates ABCG5 through the butyrate-AMPK-pHDAC5 pathway in offspring at weaning.

The role of TRPV ion channels in adipocyte differentiation: What is the evidence?

Obesity is a complex disorder, and the incidence of obesity continues to rise at an alarming rate worldwide. In particular, the growing incidence of overweight and obesity in children is a major health concern. However, the underlying mechanisms of obesity remain unclear and the efficacy of several approaches for weight loss is limited. As an important calcium-permeable temperature-sensitive cation channel, transient receptor potential vanilloid (TRPV) ion channels directly participate in thermo-, mechano-, and chemosensory responses. Modulation of TRPV ion channel activity can alter the physiological function of the ion channel, leading to neurodegenerative diseases, chronic pain, cancer, and skin disorders. In recent years, increasing studies have demonstrated that TRPV ion channels are abundantly expressed in metabolic organs, including the liver, adipose tissue, skeletal muscle, pancreas, and central nervous system, which has been implicated in various metabolic diseases, including obesity and diabetes mellitus. In addition, as an important process for the pathophysiology of adipocyte metabolism, adipocyte differentiation plays a critical role in obesity. In this review, we focus on the role of TRPV ion channels in adipocyte differentiation to broaden the ideas for prevention and control strategies for obesity.

Maternal voluntary wheel running modulates glucose homeostasis, the gut microbiota and its derived fecal metabolites in offspring.

Maternal overnutrition can dramatically increase the susceptibility of offspring to metabolic diseases, whereas maternal exercise may improve glucose metabolism in offspring. However, the underlying mechanism programming the intergenerational effects of maternal exercise on the benefits of glucose metabolism has not been fully elaborated. C57BL/6 female mice were randomly assigned to four subgroups according to a diet and exercise paradigm before and during pregnancy as follows: NC (fed with normal chow diet and sedentary), NCEx (fed with normal chow diet and running), HF (fed with high-fat diet and sedentary), and HFEx (fed with high-fat diet and running). Integrative 16S rDNA sequencing and mass spectrometry-based metabolite profiling were synchronously performed to characterize the effects of maternal exercise on the gut microbiota composition and metabolite alterations in offspring. Maternal exercise, acting as a natural pharmaceutical intervention, prevented deleterious effects on glucose metabolism in offspring. 16S rDNA sequencing revealed remarkable changes in the gut microbiota composition in offspring. Metabolic profiling indicated multiple altered metabolites, which were enriched in butanoate metabolism signaling in offspring. We further found that maternal exercise could mediate gene expression related to intestinal gluconeogenesis in offspring. In conclusion, our study indicated that maternal running significantly improved glucose metabolism in offspring and counteracted the detrimental effects of maternal high-fat feeding before and during pregnancy. We further demonstrated that maternal voluntary wheel running could integratively program the gut microbiota composition and fecal metabolite changes and then regulate butanoate metabolism and mediate intestinal gluconeogenesis in offspring.

Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259.

OBJECTIVE: Cardiomyocyte senescence is an important contributor to cardiovascular diseases and can be induced by stressors including DNA damage, oxidative stress, mitochondrial dysfunction, epigenetic regulation, etc. However, the underlying mechanisms for the development of cardiomyocyte senescence remain largely unknown. Sulfur dioxide (SO2) is produced endogenously by aspartate aminotransferase 2 (AAT2) catalysis and plays an important regulatory role in the development of cardiovascular diseases. The present study aimed to explore the effect of endogenous SO2 on cardiomyocyte senescence and the underlying molecular mechanisms. APPROACH AND RESULTS: We interestingly found a substantial reduction in the expression of AAT2 in the heart of aged mice in comparison to young mice. AAT2-knockdowned cardiomyocytes exhibited reduced SO2 content, elevated expression levels of Tp53, p21Cip/Waf, and p16INk4a, enhanced SA-ß-Gal activity, and elevated level of γ-H2AX foci. Notably, supplementation with a SO2 donor ameliorated the spontaneous senescence phenotype and DNA damage caused by AAT2 deficiency in cardiomyocytes. Mechanistically, AAT2 deficiency suppressed the sulphenylation of signal transducer and activator of transcription 3 (STAT3) facilitated its nuclear translocation and DNA-binding capacity. Conversely, a mutation in the cysteine (Cys) 259 residue of STAT3 blocked SO2-induced STAT3 sulphenylation and subsequently prevented the inhibitory effect of SO2 on STAT3-DNA-binding capacity, DNA damage, and cardiomyocyte senescence. Additionally, cardiomyocyte (cm)-specific AAT2 knockout (AAT2cmKO) mice exhibited a deterioration in cardiac function, cardiomegaly, and cardiac aging, whereas supplementation with SO2 donors mitigated the cardiac aging and remodeling phenotypes in AAT2cmKO mice. CONCLUSION: Downregulation of the endogenous SO2/AAT2 pathway is a crucial pathogenic mechanism underlying cardiomyocyte senescence. Endogenous SO2 modifies STAT3 by sulphenylating Cys259, leading to the inhibition of DNA damage and the protection against cardiomyocyte senescence.

TRPV3 facilitates lipolysis and attenuates diet-induced obesity via activation of the NRF2/FSP1 signaling axis.

Transient receptor potential vanilloid (TRPV) ion channels play a crucial role in various cellular functions by regulating intracellular Ca2+ levels and have been extensively studied in the context of several metabolic diseases. However, the regulatory effects of TRPV3 in obesity and lipolysis are not well understood. In this study, utilizing a TRPV3 gain-of-function mouse model (TRPV3G568V/G568V), we assessed the metabolic phenotype of both TRPV3G568V/G568V mice and their control littermates, which were randomly assigned to either a 12-week high-fat diet or a control diet. We investigated the potential mechanisms underlying the role of TRPV3 in restraining obesity and promoting lipolysis both in vivo and in vitro. Our findings indicate that a high-fat diet led to significant obesity, characterized by increased epididymal and inguinal white adipose tissue weight and higher fat mass. However, the gain-of-function mutation in TRPV3 appeared to counteract these adverse effects by enhancing lipolysis in visceral fat through the upregulation of the major lipolytic enzyme, adipocyte triglyceride lipase (ATGL). In vitro experiments using carvacrol, a TRPV3 agonist, demonstrated the promotion of lipolysis and antioxidation in 3T3-L1 adipocytes after TRPV3 activation. Notably, carvacrol failed to stimulate Ca2+ influx, lipolysis, and antioxidation in 3T3-L1 adipocytes treated with BAPTA-AM, a cell-permeable calcium chelator. Our results revealed that TRPV3 activation induced the action of transcriptional factor nuclear factor erythroid 2-related factor 2 (NRF2), resulting in increased expression of ferroptosis suppressor protein 1 (FSP1) and superoxide dismutase2 (SOD2). Moreover, the inhibition of NRF2 impeded carvacrol-induced lipolysis and antioxidation in 3T3-L1 adipocytes, with downregulation of ATGL, FSP1, and SOD2. In summary, our study suggests that TRPV3 promotes visceral fat lipolysis and inhibits diet-induced obesity through the activation of the NRF2/FSP1 signaling axis. We propose that TRPV3 may be a potential therapeutic target in the treatment of obesity.

Targeting Estrogen Receptor Beta Ameliorates Diminished Ovarian Reserve via Suppression of the FOXO3a/Autophagy Pathway.

Diminished ovarian reserve (DOR) refers to a decrease in the number and/or quality of oocytes, leading to infertility, poor ovarian response and adverse pregnancy outcomes. Currently, the pathogenesis of DOR is largely unknown, and the efficacy of existing therapeutic methods is limited. Therefore, in-depth exploration of the mechanism underlying DOR is highly important for identifying molecular therapeutic targets for DOR. Our study showed that estrogen receptor beta (ERß) mRNA and protein expression was upregulated in granulosa cells (GCs) from patients with DOR and in the ovaries of DOR model mice. Mechanistically, elevated ERß promotes forkhead transcription factor family 3a (FOXO3a) expression, which contributes to autophagic activation in GCs. Activation of FOXO3a/autophagy signalling leads to decreased cell proliferation and increased cell apoptosis and ultimately leads to DOR. In a cyclophosphamide (Cy)-induced DOR mouse model, treatment with PHTPP, a selective ERß antagonist, rescued fertility by restoring normal sex hormone secretion, estrus cycle duration, follicle development, oocyte quality and litter size. Taken together, these findings reveal a pathological mechanism of DOR based on ERß overexpression and identify PHTPP as a potential therapeutic agent for DOR.

A Degradable Bioelectronic Scaffold for Localized Cell Transfection toward Enhancing Wound Healing in a 3D Space.

Large skin wounds, with extensive surface area and deep vertical full-thickness involvement, can pose significant challenges in clinical settings. Traditional routes for repairing skin wounds encompass three hallmarks: 1) scab formation for hemostasis; 2) proliferation and migration of epidermal cells for wound closure; 3) proliferation, migration, and functionalization of fibroblasts and endothelial cells for dermal remodeling. However, this route face remarkable challenges to healing large wounds, usually leading to disordered structures and loss of functions in the regenerated skin, due to limited control on the transition among the three stages. In this work, an implantable bioelectronics is developed that enables the synchronization of the three stages, offering accelerated and high-quality healing of large skin wounds. The system efficiently electro-transfect local cells near the wounds, forcing cellular proliferation, while providing a 3D porous environments for synchronized migration of epidermal and dermal cells. In vivo experiments demonstrated that the system achieved synchronous progression of multiple layers within the wounds, leading to the reconstruction of a complete skin structure similar to healthy skin, which presents a new avenue for the clinical translation of large wound healing.

Efficient, High-Quality Engineering of Therapeutic Extracellular Vesicles on an Integrated Nanoplatform.

Engineered extracellular vesicles (EVs) have been recognized as important therapeutics for gene and cell therapy. To achieve clinically desired therapy, technologies for EV engineering have high demands on the efficacy in producing EVs and their qualities, which, however, remain challenging to conventional routes due to their limited control on therapeutic payload delivery, EV secretion, and extracellular microenvironments. Here, we report a nanoplatform (denoted as PURE) that enables efficient electro-transfection while stimulating cells to produce high-quality EVs carrying functional RNAs. PURE further employs an ammonium removal zone to maintain the physiological conditions of the extracellular microenvironment and an EV uptake zone that efficiently (87.1%) captures EVs in situ with porous hydrogels. The platform achieved about a 12-fold higher yield of engineered EVs and a 146-fold abundance of desired therapeutics compared to those naturally secreted from cells. The PURE-engineered miR-130a-EVs were validated for effectively upregulating the mTOR signaling pathway in both in vitro and in vivo. Their therapeutic capability was then verified by enhancing the in vitro activation of primordial follicles. In vivo applications further highlighted the therapeutic effects of miR-130a-EVs in restoring ovary function in aged mice. The PURE platform represents a strategy for the clinical translation of EV-mediated therapy.

Animal models of the placenta accreta spectrum: current status and further perspectives.

Placenta accreta spectrum disorder (PAS) is a kind of disease of placentation defined as abnormal trophoblast invasion of part or all of the placenta into the myometrium, even penetrating the uterus. Decidual deficiency, abnormal vascular remodeling in the maternal-fetal interface, and excessive invasion by extravillous trophoblast (EVT) cells contribute to its onset. However, the mechanisms and signaling pathways underlying such phenotypes are not fully understood, partly due to the lack of suitable experimental animal models. Appropriate animal models will facilitate the comprehensive and systematic elucidation of the pathogenesis of PAS. Due to the remarkably similar functional placental villous units and hemochorial placentation to humans, the current animal models of PAS are based on mice. There are various mouse models induced by uterine surgery to simulate different phenotypes of PAS, such as excessive invasion of EVT or immune disturbance at the maternal-fetal interface, which could define the pathological mechanism of PAS from the perspective of the "soil." Additionally, genetically modified mouse models could be used to study PAS, which is helpful to exploring the pathogenesis of PAS from the perspectives of both "soil" and "seed," respectively. This review details early placental development in mice, with a focus on the approaches of PAS modeling. Additionally, the strengths, limitations and the applicability of each strategy and further perspectives are summarized to provide the theoretical foundation for researchers to select appropriate animal models for various research purposes. This will help better determine the pathogenesis of PAS and even promote possible therapy.

Maternal high-calorie diet feeding programs hepatic cholesterol metabolism and Abca1 promoter methylation in the early life of offspring.

Maternal high-calorie diet feeding can dramatically increase the susceptibility of metabolic diseases in offspring. However, whether maternal high-calorie diet feeding can program hepatic cholesterol metabolism in the early life of offspring is less understood, and the epigenetic mechanisms underlying this intergenerational effect, especially during the early life of offspring, are unknown. Female C57BL/6J mice were randomly assigned to a high-calorie diet or control diet before and during gestation, and lactation. Lipid metabolism was evaluated in male offspring at weaning. Gene expressions and quantitative methylation levels of key genes associated with hepatic cholesterol metabolism were further evaluated in offspring at weaning age. We found that maternal high-calorie diet feeding resulted in higher body weight, hypercholesterolemia, elevated total cholesterol in liver homogenates, and fat deposits in the liver in offspring at weaning. For key genes that regulate cholesterol metabolism in liver, we showed lower Hmgcr and Ldlr, and higher Abca1 mRNA and protein expressions in offspring from dams fed with high-calorie diet at weaning age. We further found that maternal high-calorie diet feeding significantly decreased Abca1 methylation level in offspring, with lower methylation levels of both CpG 11 and CpG 22 sites. Interestingly, we found that Abca1 methylation level was negatively associated with hepatic Abca1 mRNA expression in offspring from dams fed with high-calorie diet and controls. However, the expressions of key genes associated with hepatic cholesterol metabolism were not significant between fetuses of dams fed with high-calorie diet and control diet. In conclusion, our results indicate that maternal high-calorie diet feeding results in aberrant lipid metabolism, including hypercholesterolemia and fat deposits in the liver of offspring as early as weaning age. Furthermore, maternal high-calorie feeding can program hepatic cholesterol metabolism and Abca1 methylation in the early life of offspring.

Endothelial Cell-Derived SO2 Controls Endothelial Cell Inflammation, Smooth Muscle Cell Proliferation, and Collagen Synthesis to Inhibit Hypoxic Pulmonary Vascular Remodelling.

Hypoxic pulmonary vascular remodelling (PVR) is the major pathological basis of aging-related chronic obstructive pulmonary disease and obstructive sleep apnea syndrome. The pulmonary artery endothelial cell (PAEC) inflammation, and pulmonary artery smooth muscle cell (PASMC) proliferation, hypertrophy and collagen remodelling are the important pathophysiological components of PVR. Endogenous sulfur dioxide (SO2) was found to be a novel gasotransmitter in the cardiovascular system with its unique biological properties. The study was aimed to investigate the role of endothelial cell- (EC-) derived SO2 in the progression of PAEC inflammation, PASMC proliferation, hypertrophy and collagen remodelling in PVR and the possible mechanisms. EC-specific aspartic aminotransferase 1 transgenic (EC-AAT1-Tg) mice were constructed in vivo. Pulmonary hypertension was induced by hypoxia. Right heart catheterization and echocardiography were used to detect mouse hemodynamic changes. Pathologic analysis was performed in the pulmonary arteries. High-performance liquid chromatography was employed to detect the SO2 content. Human PAECs (HPAECs) with lentiviruses containing AAT1 cDNA or shRNA and cocultured human PASMCs (HPASMCs) were applied in vitro. SO2 probe and enzyme-linked immunosorbent assay were used to detect the SO2 content and determine p50 activity, respectively. Hypoxia caused a significant reduction in SO2 content in the mouse lung and HPAECs and increases in right ventricular systolic pressure, pulmonary artery wall thickness, muscularization, and the expression of PAEC ICAM-1 and MCP-1 and of PASMC Ki-67, collagen I, and α-SMA (p < 0.05). However, EC-AAT1-Tg with sufficient SO2 content prevented the above increases induced by hypoxia (p < 0.05). Mechanistically, EC-derived SO2 deficiency promoted HPAEC ICAM-1 and MCP-1 and the cocultured HPASMC Ki-67 and collagen I expression, which was abolished by andrographolide, an inhibitor of p50 (p < 0.05). Meanwhile, EC-derived SO2 deficiency increased the expression of cocultured HPASMC α-SMA (p < 0.05). Taken together, these findings revealed that EC-derived SO2 inhibited p50 activation to control PAEC inflammation in an autocrine manner and PASMC proliferation, hypertrophy, and collagen synthesis in a paracrine manner, thereby inhibiting hypoxic PVR.

Over-expression of a cardiac-specific human dopamine D5 receptor mutation in mice causes a dilated cardiomyopathy through ROS over-generation by NADPH oxidase activation and Nrf2 degradation.

Dilated cardiomyopathy (DCM) is a severe disorder caused by medications or genetic mutations. D5 dopamine receptor (D5R) gene knockout (D5-/-) mice have cardiac hypertrophy and high blood pressure. To investigate the role and mechanism by which the D5R regulates cardiac function, we generated cardiac-specific human D5R F173L(hD5F173L-TG) and cardiac-specific human D5R wild-type (hD5WT-TG) transgenic mice, and H9c2 cells stably expressing hD5F173L and hD5WT. We found that cardiac-specific hD5F173L-TG mice, relative to hD5WT-TG mice, presented with DCM and increased cardiac expression of cardiac injury markers, NADPH oxidase activity, Nrf2 degradation, and activated ERK1/2/JNK pathway. H9c2-hD5F173L cells also had an increase in NADPH oxidase activity, Nrf2 degradation, and phospho-JNK (p-JNK) expression. A Nrf2 inhibitor also increased p-JNK expression in H9c2-hD5F173L cells but not in H9c2-hD5WT cells. We suggest that the D5R may play an important role in the preservation of normal heart function by inhibiting the production of reactive oxygen species, via inhibition of NADPH oxidase, Nrf2 degradation, and ERK1/2/JNK pathways.

Estrogen Replacement Regulates Vaginal Innervations in Ovariectomized Adult Virgin Rats: A Histological Study.

Background. Our previous Gräfenberg spot findings confirmed that the distal-third areas of the anterior vaginal wall bore a significantly greater number of nerves and sexual hormone may have certain degree of influence on these significant differences. However, the role of estrogen in vaginal innervations remains controversial. Methods. To investigate whether hormonal-neural interactions occur in the vagina, sixty rats were randomly divided into six groups: Sham-operated, ovariectomy, and 4 treatment groups. After 2 weeks of treatment, vaginal biopsies were prepared with hematoxylin and eosin and PGP9.5 using immunohistochemistry. Results. The density of small nerve fibers was significantly higher in the distal-half areas of intact vaginal walls than the proximal-half areas (P = 0.001). In contrast, the overall PGP 9.5-ir fiber innervation density was significantly decreased in the OVX rats subjected to surgical menopause. Sustained estrogen administration for 2 weeks resulted in nerve fiber proliferation, with values reaching normal levels in the low-dose estradiol valerate group. Conclusion. Our findings indicate that systemic hormonal therapy with low-dose estradiol valerate is effective and safe for treating deficient vaginal innervation caused by low level of estrogen activity in menopausal women and may aid studies to identify an optimal estradiol dose to provide relief from vaginal discomfort.

Differential Regulation of Morphology and Estrogen Receptor-Alpha Expression in the Vagina of Ovariectomized Adult Virgin Rats by Estrogen Replacement: A Histological Study.

Background. To determine the exact role of estrogen in vaginal tissue morphology and estrogen receptor-alpha (ERα) distribution in the vagina, which remains controversial. Methods. Sixty rats were randomly categorized: sham-operated (sham), ovariectomy (OVX), and four estradiol treatments (estradiol valerate at 0.4, 0.8, 1.6, and 3.2 mg/kg/day) for 2 weeks. Thereafter, vaginal samples were biopsied from the distal- and proximal-half portions. The percentage of ERα-immunoreactive cells and the ERα score were quantified using immunohistochemistry to assess changes in ERα expression and distribution. Results. OVX induced significant vaginal atrophy and organic index. Estrogen-replacement therapy (ERT) reversed vaginal atrophy. The vaginal distal-half areas showed lower ERα% than the proximal-half areas. The ERα% increased sharply 4 weeks after OVX, especially in the epithelial layer (P = 0.023). ERT elicited different degrees of reductions in tissues after the 2-week treatment, but the ERα% in only the epithelium recovered in parallel with that in the sham group (P = 0.001). The OVX group showed higher ERα histological scores than the sham group, and the distal-half area changed more evidently than the proximal-half area. ERα expression was nearly unchanged after ERT (P > 0.05). Conclusions. ERT is effective for treating obesity and vulvovaginal atrophy caused by hypoestrogenism and advancing age in menopausal women but cannot recover the distribution and expression of ERα.

Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility.

Skin integrity is essential for protection from external stress and trauma. Defects in structural proteins such as keratins cause skin fragility, epitomized by epidermolysis bullosa (EB), a life-threatening disorder. Here we show that dominant mutations of KLHL24, encoding a cullin 3-RBX1 ubiquitin ligase substrate receptor, cause EB. We have identified start-codon mutations in the KLHL24 gene in five patients with EB. These mutations lead to truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-ΔN28). KLHL24-ΔN28 is more stable than its wild-type counterpart owing to abolished autoubiquitination. We have further identified keratin 14 (KRT14) as a KLHL24 substrate and found that KLHL24-ΔN28 induces excessive ubiquitination and degradation of KRT14. Using a knock-in mouse model, we have confirmed that the Klhl24 mutations lead to stabilized Klhl24-ΔN28 and cause Krt14 degradation. Our findings identify a new disease-causing mechanism due to dysregulation of autoubiquitination and open new avenues for the treatment of related disorders.

Association between the SIRT1 mRNA expression and acute coronary syndrome.

AIM: Silent mating type information regulator 2 homolog 1 (SIRT1) functions as an atheroprotective factor in vascular biology, and genetic variations in SIRT1 are associated with coronary artery calcification and type 2 diabetes in several populations. In this study, we investigated the relationship between the mRNA expression levels of the SIRT1 gene and single nucleotide polymorphisms (SNPs) in the context of acute coronary syndrome (ACS). METHODS: Whole-genome expression microarray and real-time PCR techniques were used to detect the gene expression levels, and Western blotting was performed to determine the protein expression level. The four selected SNPs were genotyped in a Taqman genotyping platform. RESULTS: Compared with that observed in the controls, the mRNA expression levels of the SIRT1 gene in the microarray study were significantly lower in the acute myocardial infarction (AMI), unstable angina (UA) and overall ACS patients. These results were replicated in another independent cohort with respect to the mRNA (AMI, p＜0.001; UA, p＜0.001; ACS, p＜0.001) and protein (p＜0.05) levels. Furthermore, the relationship between the SIRT1 mRNA expression and the genotypes of four possible functional SNPs (rs12778366, rs3758391, rs2273773 and rs4746720) was tested, the results of which showed significant differences in the SIRT1 mRNA expression among the allelic genes of rs3758391 (p＜0.01) in the healthy participants. CONCLUSIONS: The present results confirm that the SIRT1 gene plays a protective role against ACS and that the rs3758391 SNP affects the mRNA expression in healthy participants, providing new insight into the processes regulating the genetic control of the SIRT1 gene with respect to the pathogenesis of ACS.

Coactivator-associated arginine methyltransferase 1 targeted by miR-15a regulates inflammation in acute coronary syndrome.

OBJECTIVE: Coactivator-associated arginine methyltransferase 1 (CARM1) is essential for the activation of a subset of NF-кB-dependent genes, which code the chemokines triggering plaque vulnerability. Unstable atherosclerotic plaques lead to the onset of acute coronary syndrome (ACS). Therefore, we aimed to investigate whether CARM1 is involved in the pathogenesis of ACS and ascertain the regulatory mechanism of CARM1 expression at posttranscriptional level. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of 19 patients with ACS and 22 subjects with risk factors for coronary heart disease. Gene expression was determined by quantitative real-time PCR and Western blot. The effects of CARM1 and miR-15a on their target genes expression were assessed by gain-of-function and loss-of-function approaches. RESULTS: PBMCs from patients with ACS showed higher levels of CARM1 mRNA and protein expression. The levels of CARM1 mRNA were positively correlated with three chemokines including interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and interleukin-8 (IL-8) in PBMCs (CARM1 and IP-10: r=0.55, P=0.008; CARM1 and MCP-1: r=0.64, P=0.002; CARM1 and IL-8: r=0.55, P=0.008). Moreover, CARM1 regulated the transcription of these chemokines in human embryonic kidney 293T (HEK293T) cells. We also found that the levels of miR-15a were decreased by 37% in patients with ACS and miR-15a modulated CARM1 expression through targeted binding to CARM1 3'-UTR. CONCLUSION: The present study demonstrated that CARM1 targeted by miR-15a played an important role in chemokine activation in the pathogenesis of ACS.

Functional analysis of single-nucleotide polymorphisms in the regulation of coactivator-associated arginine methyltransferase 1 expression and plasma homocysteine levels.

BACKGROUND: Hyperhomocysteinemia is a risk factor for cardiovascular disease. Coactivator-associated arginine methyltransferase 1 (CARM1) participates in the synthesis of homocysteine, but whether the genetic variations regulate CARM1 expression and homocysteine levels remains unknown. METHODS AND RESULTS: Functional analyses combined with an association study were conducted to identify the causal variant for CARM1 expression and homocysteine levels. Based on functional annotations obtained from Encyclopedia of DNA Elements, we selected 4 potentially functional single-nucleotide polymorphisms in the CARM1 gene and investigated their effect on CARM1 transcription levels in vivo. rs117569851, located in the promoter region of CARM1, as well as rs12460421 and rs4804544, was associated with CARM1 expression levels, and the last 2 single-nucleotide polymorphisms were discovered in high linkage disequilibrium with rs117569851 (r(2)=0.9 and 1.0) in our study sample. rs117569851 was further identified to be responsible for regulating CARM1 expression. The T allele disrupted the binding of early growth response-1, which led to the downregulation of transcriptional activity in vitro and CARM1 mRNA levels in vivo. In addition, rs117569851 was associated with plasma homocysteine levels in a Chinese population (n=406), with a 2.16 µmol/L decrease per copy of T allele. CONCLUSIONS: The present study suggests that a noncoding variant in the CARM1-promoter functions as a regulator of gene transcription and homocysteine levels.