Primary Ciliogenesis by 2-Isopropylmalic Acid Prevents PM2.5-Induced Inflammatory Response and MMP-1 Activation in Human Dermal Fibroblasts and a 3-D-Skin Model

Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.

METTL3-Induced miR-222-3p Upregulation Inhibits STK4 and Promotes the Malignant Behaviors of Thyroid Carcinoma Cells

Context Abnormally high expression of N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) has been implied to accompany thyroid carcinoma (TC) development. Objective This study aimed to explore the protumorigenic role and downstream signaling axis of METTL3 in TC. Methods This study was conducted at the Sun Yat-Sen Memorial Hospital Sun Yat-Sen University. METTL3 and miR-222-3p were overexpressed or downregulated in TC cells. Tumor and adjacent normal tissues were collected from 80 patients (19 men and 60 women, aged 30-70 years) with a pathological diagnosis of TC from January 2012 to January 2015. Cells were classified and subjected to different treatments. The expression of METTL3 was validated in TC tissues and cell lines. In functional studies, METTL3 and miR-222-3p were overexpressed or downregulated in TC cells to evaluate their effects on malignant behaviors, which were subsequently verified by xenografts in nude mice. Results The expression of METTL3 was elevated in TC, correlating with poor prognosis of TC patients. Heightened METTL3 expression accelerated malignant behaviors of TC cells. Mechanistically, METTL3 stimulated miR-222-3p expression by mediating the m6A modification of pri-miR-222-3p. miR-222-3p targeted and inversely regulated serine/threonine stress kinase 4 (STK4). Knockdown of METTL3 augmented STK4 expression by downregulating miR-222-3p, thereby suppressing the malignant behaviors of TC cells as well as tumor growth and lung metastasis in nude mice. Conclusion Silencing METTL3 suppresses miR-222-3p expression and thus stimulates STK4 expression, thereby repressing the malignancy and metastasis of TC.

2-IPMA Ameliorates PM2.5-Induced Inflammation by Promoting Primary Ciliogenesis in RPE Cells

Primary cilia mediate the interactions between cells and external stresses. Thus, dysregulation of primary cilia is implicated in various ciliopathies, e.g., degeneration of the retina caused by dysregulation of the photoreceptor primary cilium. Particulate matter (PM) can cause epithelium injury and endothelial dysfunction by increasing oxidative stress and inflammatory responses. Previously, we showed that PM disrupts the formation of primary cilia in retinal pigment epithelium (RPE) cells. In the present study, we identified 2-isopropylmalic acid (2-IPMA) as a novel inducer of primary ciliogenesis from a metabolite library screening. Both ciliated cells and primary cilium length were increased in 2-IPMA-treated RPE cells. Notably, 2-IPMA strongly promoted primary ciliogenesis and restored PM2.5-induced dysgenesis of primary cilia in RPE cells. Both excessive reactive oxygen species (ROS) generation and activation of a stress kinase, JNK, by PM2.5 were reduced by 2-IPMA. Moreover, 2-IPMA inhibited proinflammatory cytokine production, i.e., IL-6 and TNF-α, induced by PM2.5 in RPE cells. Taken together, our data suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in RPE cells.

Cadmium, an Environmental Contaminant, Exacerbates Alzheimer’s Pathology in the Aged Mice’s Brain

Cadmium (Cd) is an environmental contaminant, which is a potential risk factor in the progression of aging-associated neurodegenerative diseases. Herein, we have assessed the effects of chronic administration of Cd on cellular oxidative stress and its associated Alzheimer’s disease (AD) pathologies in animal models. Two groups of mice were used, one group administered with saline and the other with Cd (1 mg/kg/day; intraperitoneally) for 3 months. After behavioral studies, molecular/biochemical (Immunoblotting, ELISAs, ROS, LPO, and GSH assays) and morphological analyses were performed. We observed an exacerbation of memory and synaptic deficits in chronic Cd-injected mice. Subacute and chronic Cd escalated reactive oxygen species (ROS), suppressed the master antioxidant enzymes, e.g., nuclear factor-erythroid 2-related factor 2 and heme oxygenase-1, and evoked the stress kinase phospho-c-Jun N-terminal kinase 1 signaling pathways, which may escalate AD pathologies possibly associated with amyloidogenic processes. These findings suggest the regulation of oxidative stress/ROS and its associated amyloid beta pathologies for targeting the Cd-exacerbated AD pathogenesis. In addition, these preclinical animal studies represent a paradigm for epidemiological studies of the human population exposed to chronic and subacute administration of Cd, suggesting avoiding environmental contaminants.

Stress-driven cardiac calcium mishandling via a kinase-to-kinase crosstalk

Calcium homeostasis in the cardiomyocyte is critical to the regulation of normal cardiac function. Abnormal calcium dynamics such as altered uptake by the sarcoplasmic reticulum (SR) Ca2+-ATPase and increased diastolic SR calcium leak are involved in the development of maladaptive cardiac remodeling under pathological conditions. Ca2+/calmodulin-dependent protein kinase II-δ (CaMKIIδ) is a well-recognized key molecule in calcium dysregulation in cardiomyocytes. Elevated cellular stress is known as a common feature during pathological remodeling, and c-jun N-terminal kinase (JNK) is an important stress kinase that is activated in response to intrinsic and extrinsic stress stimuli. Our lab recently identified specific actions of JNK isoform 2 (JNK2) in CaMKIIδ expression, activation, and CaMKIIδ-dependent SR Ca2+ mishandling in the stressed heart. This review focuses on the current understanding of cardiac SR calcium handling under physiological and pathological conditions as well as the newly identified contribution of the stress kinase JNK2 in CaMKIIδ-dependent SR Ca2+ abnormal mishandling. The new findings identifying dual roles of JNK2 in CaMKIIδ expression and activation are also discussed in this review.

JNK2, A Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca 2+ ] SR Leak-Load Relationship

Rationale: We recently discovered pivotal contributions of stress kinase JNK2 in increased risk of atrial fibrillation (AF) through enhanced diastolic sarcoplasmic reticulum (SR) Ca 2+ leak via ryanodine receptors (RyR2). However, the role of JNK2 in the function of the SR Ca 2+ -ATPase (SERCA2), essential in maintaining [Ca 2+ ] SR cycling during each heartbeat, is completely unknown. Objective: To test the hypothesis that JNK2 increases SERCA2 activity [Ca 2+ ] SR and exacerbates an arrhythmic [Ca 2+ ] SR leak-load relationship. Methods and Results: We used confocal Ca 2+ imaging in myocytes and HEK cells, biochemistry, dual Ca 2+ /voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1, increased SERCA2 uptake and consequently elevated [Ca 2+ ]SR load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased Vmax, without altering Ca 2+ affinity (Km). Unlike the CaMKII-dependent JNK2 action in SR Ca 2+ leak, JNK2-driven SERCA2 function was CaMKII-independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca 2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca 2+ leak present, the JNK2-enhanced SR Ca 2+ uptake limited leak-induced reduction in SR Ca 2+ , normalizing Ca 2+ transient amplitude, but at a higher arrhythmogenic SR Ca 2+ leak. JNK2-specific inhibition completely normalized SR Ca 2+ handling, attenuated arrhythmic Ca 2+ activities, and alleviated AF susceptibility in aged and alcohol-exposed myocytes and intact hearts. Conclusions: We have identified a novel JNK2-induced activation of SERCA2. The dual-action of JNK2 in CaMKII-dependent arrhythmic SR Ca 2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca 2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for AF prevention and treatment.

Dietary α-Linolenic Acid Counters Cardioprotective Dysfunction in Diabetic Mice: Unconventional PUFA Protection

Whether dietary omega-3 (n-3) polyunsaturated fatty acid (PUFA) confers cardiac benefit in cardiometabolic disorders is unclear. We test whether dietary α-linolenic acid (ALA) enhances myocardial resistance to ischemia-reperfusion (I-R) and responses to ischemic preconditioning (IPC) in type 2 diabetes (T2D); and involvement of conventional PUFA-dependent mechanisms (caveolins/cavins, kinase signaling, mitochondrial function, and inflammation). Eight-week male C57Bl/6 mice received streptozotocin (75 mg/kg) and 21 weeks high-fat/high-carbohydrate feeding. Half received ALA over six weeks. Responses to I-R/IPC were assessed in perfused hearts. Localization and expression of caveolins/cavins, protein kinase B (AKT), and glycogen synthase kinase-3β (GSK3β); mitochondrial function; and inflammatory mediators were assessed. ALA reduced circulating leptin, without affecting body weight, glycemic dysfunction, or cholesterol. While I-R tolerance was unaltered, paradoxical injury with IPC was reversed to cardioprotection with ALA. However, post-ischemic apoptosis (nucleosome content) appeared unchanged. Benefit was not associated with shifts in localization or expression of caveolins/cavins, p-AKT, p-GSK3β, or mitochondrial function. Despite mixed inflammatory mediator changes, tumor necrosis factor-a (TNF-a) was markedly reduced. Data collectively reveal a novel impact of ALA on cardioprotective dysfunction in T2D mice, unrelated to caveolins/cavins, mitochondrial, or stress kinase modulation. Although evidence suggests inflammatory involvement, the basis of this “un-conventional” protection remains to be identified.