Influence of Redox Stress on Crosstalk between Fibroblasts and Keratinocytes

Although the skin is constantly subjected to endogenous and exogenous stress, it maintains a homeostatic state through wound repair and regeneration pathways. Treatment for skin diseases and injury requires a significant understanding of the various mechanisms and interactions that occur within skin cells. Keratinocytes and fibroblasts interact with each other and act as key players in the repair process. Although fibroblasts and keratinocytes are widely studied in wound healing and skin remodeling under different conditions, the influence of redox stress on keratinocyte-fibroblast crosstalk has not been thoroughly investigated. In this study, we used cold atmospheric plasma (CAP) to generate and deliver oxidative stress to keratinocytes and fibroblasts and to assess its impact on their interactions. To this end, we used a well-established in vitro 3D co-culture model imitating a realistic scenario. Our study shows that low CAP exposure is biocompatible and does not affect the viability or energetics of fibroblasts and keratinocytes. Exposure to low doses of CAP enhanced the proliferation rate of cells and stimulated the expression of key genes (KGF, MMP2, GMCSF, IL-6, and IL-8) in fibroblasts, indicating the activation and initiation of the skin repair process. Additionally, enhanced migration was observed under co-culture conditions under the given redox stress conditions, and expression of the upstream regulator and the effectors of the Hippo pathway (YAP and CYR61, respectively), which are associated with enhanced migration, were elevated. Overall, this study reinforces the application of CAP and redox stress in skin repair physiology.

The Human Positive Cofactor 4 is a Promising Chemotherapeutic Target in Lung Adenocarcinoma

Reduced sensitivity to chemotherapeutic drugs is almost inevitable in lung adenocarcinoma patients. Thus, understanding the relevant mechanisms is urgent. Positive cofactor 4 (PC4) was at first revealed to be a coactivator of basal transcription. Previous research has shown that PC4 participates in various cellular processes in normal and malignant cells. However, it is still unknown whether PC4 participates in altering the lung adenocarcinoma cell sensitivity to chemotherapy, and the relevant mechanisms remain to be explained. In this study, we discovered that PC4 was overexpressed in cisplatin-resistant lung adenocarcinoma cells. PC4 decreased cisplatin’s cytotoxic effects on lung adenocarcinoma in vivo and in vitro. Furthermore, PC4 positively correlated with SOX9 in multiple cancers. PC4 was an upstream regulator of SOX9 in lung adenocarcinoma. Furthermore, PC4 mediated lung adenocarcinoma cell sensitivity to the HIF-PH inhibitor DMOG and the mTOR inhibitor rapamycin, and PC4 mediated the synergistic effect of DMOG and cisplatin. Finally, PC4 destabilized HIF-1α upon cisplatin treatment. Our research showed that PC4 participates in mediating lung adenocarcinoma cell sensitivity to multiple drugs. Mechanistically, PC4 governs multiple downstream pathways associated with chemotherapy resistance, including the SOX9 and HIF-1α pathways. Thus, PC4 is a promising chemotherapeutic target in lung adenocarcinoma.

MiRNA-132/212 regulates tight junction stabilization in blood–brain barrier after stroke

MicroRNA-132/212 has been supposed as a critical gene related to the blood–brain barrier (BBB) protection after stroke, but its regulation pathway including the upstream regulator and downstream targets is still unclear. Herein, we demonstrated the cAMP response element-binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) to be the upstream regulator of miRNA-132/212 using CRTC1 knockout and wild-type mice. CRTC1 deletion led to the reduction of miRNA-132/212 expression in mice brain after ischemic stroke, significantly increased infarct volume, and aggravated BBB permeability with worsening neurological deficits. Furthermore, we identified that miRNA-132 repressed Claudin-1, tight junction-associated protein-1 (TJAP-1), and RNA-binding Fox-1 (RBFox-1) by directly binding to their respective 3′-untranslated regions, which alleviated the ischemic damage by enhancing neuronal survival and BBB integrity. Moreover, the co-culture of endothelial cells with CRTC1-deficient neurons aggravated the cell vulnerability to hypoxia, also supporting the idea that miRNA-132/212 cluster is regulated by CRTC1 and acts as a crucial role in the mitigation of ischemic damage. This work is a step forward for understanding the role of miRNA-132/212 in neurovascular interaction and may be helpful for potential gene therapy of ischemic stroke.

Regulation of MDM2 E3 ligase-dependent vascular calcification by MSX1/2

Vascular calcification increases morbidity and mortality in patients with cardiovascular and renal diseases. Previously, we reported that histone deacetylase 1 prevents vascular calcification, whereas its E3 ligase, mouse double minute 2 homolog (MDM2), induces vascular calcification. In the present study, we identified the upstream regulator of MDM2. By utilizing cellular models and transgenic mice, we confirmed that E3 ligase activity is required for vascular calcification. By promoter analysis, we found that both msh homeobox 1 (Msx1) and msh homeobox 2 (Msx2) bound to the MDM2 promoter region, which resulted in transcriptional activation of MDM2. The expression levels of both Msx1 and Msx2 were increased in mouse models of vascular calcification and in calcified human coronary arteries. Msx1 and Msx2 potentiated vascular calcification in cellular and mouse models in an MDM2-dependent manner. Our results establish a novel role for MSX1/MSX2 in the transcriptional activation of MDM2 and the resultant increase in MDM2 E3 ligase activity during vascular calcification.

Collaborations Involving Malaysia Upstream Regulator to Enhance Decommissioning

This paper aims to share the collaboration efforts of Malaysia's governing body for Upstream oil & gas industry with various stakeholders to enhance decommissioning in Malaysia. By virtue of Section of the Petroleum Development Act 1974, Petroliam Nasional Berhad (PETRONAS) ("PETRONAS") is vested with the entire ownership in, and the exclusive rights, powers, liberties and privileges of exploring, exploiting, winning and obtaining petroleum lying onshore or offshore of Malaysia. MPM manages the decommissioning liabilities for all Upstream petroleum facilities in Malaysia, specifically to strategize, regulate, promote, and drive decommissioning execution that is safe, cost-effective and brings the best benefit to the environment. Due to the shift in the industry with uncertainties in the long-term crude oil prices, depleting reserves, and operating cost challenges, it has made this non-revenue generating activity unavoidable. Thus, it is crucial to drive down decommissioning costs while protecting the environment. To achieve this objective, one of the focused initiatives pursued by MPM is through collaborations with relevant stakeholders such as industry players, upstream operators, government bodies, and academia. Such collaborations were found to be the fastest way to develop innovative solutions whereby the collaborators work together to achieve a common goal. Collaborations were done through, among others, constant and systematic engagements, workshops, brainstorming sessions, etc. A notable example would be in 2017, MPM successfully entered into a Memorandum of Understanding ("MOUs") with the Department of Fisheries ("DOF"), Ministry of Agriculture and Fishery Industry on rigs-to-reef. In 2020, MPM had successfully conducted a series of virtual workshops to capture decommissioning enhancement areas and lessons learnt with Operators and decommissioning contractors based on real-life experiences and past projects in Malaysia. A total of 115 enhancements areas were captured for consideration. Beyond these two items, MPM had successfully collaborated with many other stakeholders related to decommissioning and will continue to explore more collaborations in the future to support decommissioning in Malaysia. Details of these collaborations will be shared as part of this presentation. There were great experiences and important lessons that PETRONAS had learnt from these collaborations. PETRONAS believes that the culture of sharing experiences and lessons learnt will be the epitome for Operators and Contractors to work safely, stimulate creativity and strive towards decommissioning cost compression for every decommissioning project.

Genetic Regulation of RIPK1 and Necroptosis

The receptor-interacting protein kinase 1 (RIPK1) is recognized as a master upstream regulator that controls cell survival and inflammatory signaling as well as multiple cell death pathways, including apoptosis and necroptosis. The activation of RIPK1 kinase is extensively modulated by ubiquitination and phosphorylation, which are mediated by multiple factors that also control the activation of the NF-κB pathway. We discuss current findings regarding the genetic modulation of RIPK1 that controls its activation and interaction with downstream mediators, such as caspase-8 and RIPK3, to promote apoptosis and necroptosis. We also address genetic autoinflammatory human conditions that involve abnormal activation of RIPK1. Leveraging these new genetic and mechanistic insights, we postulate how an improved understanding of RIPK1 biology may support the development of therapeutics that target RIPK1 for the treatment of human inflammatory and neurodegenerative diseases.

Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.

Investigation of Ifosfamide Toxicity Induces Common Upstream Regulator in Liver and Kidney

Ifosfamide is an alkylating agent, a synthetic analogue of cyclophosphamide, used to treat various solid cancers. In this study, the toxicity of ifosfamide was evaluated using single-and multiple-dose intraperitoneal administration in rats under Good Laboratory Practice guidelines, and an additional microarray experiment was followed to support toxicological findings. A single dose of ifosfamide (50 mg/kg) did not induce any pathological changes. Meanwhile, severe renal toxicity was observed in the 7 and 28 days consecutively administered groups, with significant increases in blood urea nitrogen and creatinine levels. In the tox-list analysis, cholesterol synthesis-related genes were mostly affected in the liver and renal failure-related genes were affected in the kidney after ifosfamide administration. Moreover, interferon regulatory factor 7 was selected as the main upstream regulator that changed in both the liver and kidney, and was found to interact with other target genes, such as ubiquitin specific peptidase 18, radical S-adenosyl methionine domain containing 2, and interferon-stimulated gene 15, which was further confirmed by real-time RT-PCR analysis. In conclusion, we confirmed kidney-biased ifosfamide organ toxicity and identified identically altered genes in both the liver and kidney. Further comprehensive toxicogenomic studies are required to reveal the exact relationship between ifosfamide-induced genes and organ toxicity.