First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Reviving human γδT cells from apoptosis induced by IL-12/18 via p-JNK inhibition.

γδT cells recognize and exert cytotoxicity against tumor cells independently of MHC restriction and have antigen presentation and regulatory functions to promote adaptive immune responses. They are considered as potential immune cells for cellular immunotherapy in cancer patients. However, it is challenging to ex vivo expand human γδT cells that have superb effector functions and long-term survival for adoptive cancer therapy. We found that IL-12/18 combination could drastically promote IFN-γ secretion and cytotoxicity in human γδT cells. However, the enhanced activation of human γδT cells is accompanied by increased apoptosis and elevated expressions of co-inhibitory receptors under the stimulation of IL-12/18. We further demonstrated that IL-12/18 induced apoptosis of human γδT cells was in a phosphoantigen or IFN-γ-independent manner. Transcriptomic analysis suggested that IL-12/18-induced apoptosis of human γδT cells was mediated by the activation of JNK pathway. p-JNK inhibitor (SP-600125) treatment effectively revived human γδT cells from the apoptosis induced by IL-12/18 and maintained their enhanced IFN-γ production and cytotoxicity against tumor cells. Our results provide a novel and feasible strategy for ex vivo expansion of cytokine-activated human γδT cells, which could promote the efficacy of γδT cell adoptive immunotherapy in cancer patients.

Restoration of erectile function by a combination of antiapoptosis by JNK inhibitor and preservation of smooth muscle or endothelium by hepatocyte growth factor in a rat model of cavernous nerve injury.

BACKGROUND: Because of structural alterations in the corpus cavernosum after radical prostatectomy (RP), post-RP erectile dysfunction remains a very difficult condition to treat. We aimed to determine if the combined administration of a Jun-amino terminal kinase (JNK) inhibitor and hepatocyte growth factor (HGF) in the immediate post-injury period would restore erectile function by antiapoptotic and pro-regenerative effects through the rectification of molecular pathways related to the structural integrity of the penis in a rat model of bilateral cavernosal nerve crush injury (CNCI). METHODS: A total of 70 rats were divided into five groups: Sham surgery (S), CNCI (I), and once-daily intraperitoneal administration of 10.0 mg/kg JNK inhibitor + twice-weekly intracavernosal administration of low-dose (2.1 µg), medium-dose (4.2 µg), or high-dose (8.4 µg) HGF (I + J + LH or I + J + MH or I + J + HH, respectively) in the immediate post-injury period. Erectile responses to electrostimulation (1.0, 3.0, and 5.0 V), histological staining, caspase-3 activity, and Western blotting were evaluated 9 days after surgery. RESULTS: Group I showed lower intracavernosal pressure (ICP)/mean arterial pressure (MAP) after stimulation at each voltage, lower area under the curve (AUC)/MAP after stimulation at each voltage, less smooth muscle (SM) content, a lower SM/collagen ratio, higher caspase-3 activity, increased cJun phosphorylation, decreased protein expression of PECAM-1, decreased cMet phosphorylation, and decreased endothelial nitric oxide synthase (eNOS) phosphorylation compared to Group S. The SM content, SM/collagen ratio, protein expression of ICP/MAP, or AUC/MAP after stimulation at each voltage in Group I + J + LH were partially restored, despite the normalization of cJun phosphorylation and caspase-3 activity. The ICP/MAP, AUC/MAP, caspase-3 activity, SM content, protein expression of PECAM-1, cJun phosphorylation, cMet phosphorylation, and eNOS phosphorylation in both Groups I + J + MH and I + J + HH were restored to the levels observed in Group S, while the SM/collagen ratio was significantly improved but not completely normalized. CONCLUSIONS: Our data indicated that the combined administration of a JNK inhibitor and medium or high-dose HGF to nerve-injured rats in the immediate post-injury period after CNCI may restore erectile function to a level comparable to the normal level by suppressing cavernosal apoptosis and preserving the integrity of SM or endothelium via rectification of the cJun and cMet/eNOS pathways.

Combination Therapy with a JNK Inhibitor and Hepatocyte Growth Factor for Restoration of Erectile Function in a Rat Model of Cavernosal Nerve Injury: Comparison with a JNK Inhibitor Alone or Hepatocyte Growth Factor Alone.

We determined if combined administration of JNK-inhibitors and HGF (hepatocyte-growth-factor) would restore erectile-function through both antiapoptotic and regenerative effects in a rat model of cavernous-nerve-crush-injury (CNCI), and compared the results with administration of JNK-inhibitor alone or HGF alone. We randomized 70 rats into 5 groups: sham-surgery-group (S), CNCI (I) group, a group treated with once-daily intraperitoneal-administration of 10.0-mg/kg of JNK-inhibitors (J), a twice-weekly intracavernosal-administration of 4.2-µg HGF group (H), and a combined-treatment with 10.0-mg/kg JNK-inhibitors and 4.2-µg HGF group (J+H). We investigated erectile-responses to electrostimulation, histological-staining, caspase-3-activity-assay, and immunoblotting at two-weeks postoperatively. The three treatment groups showed improvements in erectile-responses (ICP/MAP and AUC/MAP ratios) compared to Group-I. The erectile-responses in Group-J+H were greater than those in Group-J or Group-H. The erectile-responses in Group-J+H were generally normalized. Caspase-3-activity and cJun-phosphorylation in Group-J and Group-J+H improved compared to Group-I, whereas caspase-3-activity in Group-H partially improved. Protein-expression of PECAM-1, eNOS-phosphorylation, and smooth-muscle content in Group-J+H were normalized, although those in Group-J or Group-H were partially restored. Combination therapy with JNK-inhibitors and HGF can generally normalize erectile-function through anti-apoptosis and preservation of endothelium or SM in rat CNCI model. The combined treatment appears to be superior to the respective agent alone in terms of therapeutic effects.

Dichloroacetate enhances the anti-tumor effect of sorafenib via modulating the ROS-JNK-Mcl-1 pathway in liver cancer cells.

Liver cancer is one of the most common and high recurrence malignancies. Besides radiotherapy and surgery, chemotherapy also plays an essential role in the treatment of liver cancer. Sorafenib and sorafenib-based combination therapies have been proven efficacy against tumors. However, previous clinical studies have indicated that some patients with liver cancer are resistant to sorafenib treatment and the existing strategies are not satisfactory in the clinic. Therefore, it is urgent to investigate strategies to improve the effectiveness of sorafenib for liver cancer and to explore effective drug combinations. In the present study, we found that dichloroacetate (DCA) could significantly enhance the anti-tumor effect of sorafenib on liver cancer cells, including reduced viability and dramatically promoted apoptosis in liver cancer cells. Moreover, compared to sorafenib alone, the combination of DCA and sorafenib markedly increased the degradation of anti-apoptotic protein Mcl-1 by enhancing its phosphorylation. Overexpression of Mcl-1 could significantly attenuate the synergetic effect of DCA and sorafenib on apoptosis induction in liver cancer cells. Furthermore, we found that the ROS-JNK pathway was obviously activated in the DCA combined sorafenib group. The levels of ROS and p-JNK were dramatically up-regulated in the two drug combination groups. Antioxidant NAC could alleviate the synergetic effects of DCA and sorafenib on ROS generation, JNK activation, Mcl-1 degradation, and cell apoptosis. Moreover, DCA and sorafenib's effects on Mcl-1 degradation and apoptosis could also be inhibited by JNK inhibitor 'SP'600125. Finally, the synergetic effects of DCA and sorafenib on tumor growth suppression, Mcl-1 degradation and induction of apoptosis were also validated in liver cancer xenograft in vivo. These findings indicate that DCA enhances the anti-tumor effect of sorafenib via the ROS-JNK-Mcl-1 pathway in liver cancer cells. This study may provide new insights to improve the chemotherapeutic effect of sorafenib, which may be beneficial for further clinical application of sorafenib in liver cancer treatment.

Role of miR-27a in the regulation of cellular function via the inhibition of MAP2K4 in patients with asthma.

Asthma is a respiratory disease with a clinically high incidence, and repeated attacks of asthma severely affect the quality of life and even pose a threat to health, leading to severe burdens on families and even the society. A thorough understanding of the pathogenesis of asthma is essential for the prevention and treatment of asthma. This study aimed to examine the effect of the microRNA miR-27a on asthma and its relationship with mitogen activated protein kinase 4 (MAP2K4). Patients with asthma admitted to our hospital from August 2016 to August 2018 and healthy participants in the same period were included in this prospective analysis. The mRNA expression levels of miR-27a and MAP2K4 in peripheral blood were determined. Airway smooth muscle cells (ASMCs) were used to study the effects of miR-27a and MAP2K4 on cell biological behavior. The relationship between miR-27a and MAP2K4 was verified using dual-luciferase reporter assay. miR-27a expression was increased and MAP2K4 mRNA expression was decreased in asthma (P < 0.05). Increasing miR-27a expression and inhibiting MAP2K4 expression could enhance the activity of ASMCs, whereas inhibiting miR-27a expression and increasing MAP2K4 expression had the opposite effect (P < 0.05). Dual-luciferase reporter assay results showed that the fluorescence activity of MAP2K4-wild type was inhibited by increased miR-27a expression (P < 0.05). miR-27a promotes the proliferation and invasion of ASMCs by targeting MAP2K4 and is involved in the occurrence of asthma.

Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

A novel indazole derivative, compound Cyy-272, attenuates LPS-induced acute lung injury by inhibiting JNK phosphorylation.

Acute lung injury (ALI) is a diffuse lung dysfunction disease characterized by high prevalence and high mortality. Thus far, no effective pharmacological treatment has been made for ALI in clinics. Inflammation is critical to the development of ALI. Therefore, anti-inflammation may be a potential therapy strategy for ALI. Indazole-containing derivatives, representing one of the most important heterocycles in drug molecules, are endowed with a broad range of biological properties, such as anti-cancer and anti-inflammation. In the current study, we investigated the biological effects of Cyy-272, a newly synthesized indazole compound, on LPS-induced ALI both in vivo and in vitro. Results show that Cyy-272 can inhibit the release of inflammatory cytokines in LPS-stimulated macrophage and alleviate LPS induced ALI. Further experiment revealed that Cyy-272 exhibit anti-inflammation activity by inhibiting JNK phosphorylation. Overall, our studies show that an indazole derivative, Cyy-272, is effective in suppressing LPS-induced JNK activation and inflammatory signaling.

Conophylline Suppresses Angiotensin II-Induced Myocardial Fibrosis In Vitro via the BMP4/JNK Pathway.

We studied the effects and mechanisms of action of conophylline in different concentrations in the original in vitro model of myocardial fibrosis (treatment of cardiac fibroblasts isolated form the hearts of newborn rats with angiotensin II). Viability, collagen content, and expression of related protein in cardiac fibroblasts were assessed using the MTT-test, Sircol assay, and Western blotting, respectively. Conophylline markedly protected the cultured cells against the development of angiotensin II-induced fibrosis, which was seen from reduced viability of fibroblasts, decreased collagen content, and down-regulation of the expression of α-smooth muscle actin (α-SMA). Conophylline did not affect the TGF-ß pathway altered by angiotensin II, but markedly decreased the level of bone morphogenetic protein-4 (BMP4) enhanced by angiotensin II and BMP4 itself. Conophylline produced no effect on phosphorylation of α-SMA and Smad homologue-1/5/8, the classic BMP4 downstream pathway elements, but reduced the level of c-Jun N-terminal kinase (JNK) elevated by BMP4. Conophylline did not inhibit the development of myocardial fibrosis in the presence of JNK activator anisomycin. Thus, conophylline inhibited angiotensin II-provoked myocardial fibrosis via the BMP4/JNK pathway.

JNK inhibition alleviates delayed neurocognitive recovery after surgery by limiting microglia pyroptosis.

Delayed neurocognitive recovery (dNCR) is a prevalent complication after surgery in older adults. Neuroinflammation plays a pivotal role in the pathogenesis of dNCR. Recently,compelling evidence suggests that theinvolvement of microglia pyroptosis in the regulation of neuroinflammation in neurologicaldiseases. Nevertheless, the exact role of microglia pyroptosis in dNCR remains elusive. In the study, in vitro and in vivo models of dNCR were used to examine the potential effects of the mitogen­activated protein kinase signaling pathway on Nod-like receptor protein 3 (NLRP3) inflammasome-mediated microglia pyroptosis and cognitive deficits following surgery. In vivo, we observed surgery-induced upregulation of phosphorylated (p)-c-Jun N-terminal kinases (JNK) in microglia and subsequently NLRP3 inflammasome activation, pyroptosis, and inflammatory cytokines release in mice hippocampus. Interestingly, JNK inhibitor SP600125 significantly attenuated surgery-induced cognitive impairments through inhibiting pyroptosis, inflammatory responses, and reducing immunoreactivity of NLRP3 and gasdermin D N terminus (GSDMD-N) in hippocampal microglia. In vitro, NLRP3 inflammasome- and pyroptosis-associated proteins and immunoreactivity of NLRP3, GSDMD-N, and interleukin-1ß were activated in BV2 microglial cells following lipopolysaccharide (LPS) stimulation. These effects were significantly suppressed in BV2 cells by SP600125 treatment. Furthermore, treatment with NLRP3 specific inhibitor, MCC950, attenuated microglia pyroptosis induced by LPS, but did not rescue LPS-induced increased expression of p-JNK. These results indicate that the JNK pathway is largely upstream of the NLRP3 inflammasome, which exerts a crucial regulatory impact on microglia pyroptosis and inflammatory responses, thus providing a promising avenue to prevent dNCR.

Design and synthesis of 1H-pyrazolo[3,4-b]pyridines targeting mitogen-activated protein kinase kinase 4 (MKK4) - A promising target for liver regeneration.

Currently, the therapeutic options for treatment of liver failure are very limited. As mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified by in vivo RNAi experiments to be a major regulator in hepatocyte regeneration, we pursued the development of a small molecule targeting this protein kinase. Starting from the approved BRAFV600E inhibitor vemurafenib (8), that showed a high off-target affinity to MKK4 in an initial screening, we followed a scaffold-hopping approach, changing the core heterocycle from 1H-pyrrolo[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine (10). Affinity to MKK4 could be conserved while the selectivity against off-target protein kinases was slightly improved. Further modifications led to 58 and 59 showing high affinity to MKK4 in the low nanomolar range and excellent selectivity profile from mandatory multiparameter-optimization for the essential anti-targets (MKK7, JNK1) and off-targets (BRAF, MAP4K5, ZAK) in the MKK4 pathway. Herein we report the first selective MKK4 inhibitors in this class.

Eomesodermin in CD4+T cells is essential for Ginkgolide K ameliorating disease progression in experimental autoimmune encephalomyelitis.

Eomesodermin (Eomes), a transcription factor, could suppress the Th17 cell differentiation and proliferation through directly binding to the promoter zone of the Rorc and Il17a gene, meanwhile the expression of Eomes is suppressed when c-Jun directly binds to its promoter zone. Ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) is a diterpene lactone isolated from the leaves of Ginkgo biloba. A previous study indicated that GK could decrease the level of phospho JNK (c-Jun N-terminal kinase). Here, we reported the therapeutic potential of Ginkgolide K (GK) treatment to ameliorate experimental autoimmune encephalomyelitis (EAE) disease progression. Methods: EAE was induced in both wildtype and CD4-Eomes conditional knockout mice. GK was injected intraperitoneally. Disease severity, inflammation, and tissue damage were assessed by clinical evaluation, flow cytometry of mononuclear cells (MNCs), and histopathological evaluation. Dual-luciferase reporter assays were performed to measure Eomes transcription activity in vitro. The potency of GK (IC50) was determined using JNK1 Kinase Enzyme System. Results: We revealed that GK could ameliorate EAE disease progression by the inhibition of the Th17 cells. Further mechanism studies demonstrated that the level of phospho JNK was decreased and the level of Eomes in CD4+T cells was dramatically increased. This therapeutic effect of GK was almost completely interrupted in CD4-Eomes conditional knockout mice. Conclusions: These results provided the therapeutic potential of GK treatment in EAE, and further suggested that Eomes expression in CD4+T cells might be essential in this process.

Activated Beta-Catenin Signaling Ameliorates Radiation-Induced Skin Injury by Suppressing Marvel D3 Expression.

Radiation-induced skin injury remains a serious concern for cancer radiotherapy, radiation accidents and occupational exposure, and the damage mainly occurs due to apoptosis and reactive oxygen species (ROS) generation. There is currently no effective treatment for this disorder. The ß-catenin signaling pathway is involved in the repair and regeneration of injured tissues. However, the role of the ß-catenin signaling pathway in radiation-induced skin injury has not been reported. In this study, we demonstrated that the ß-catenin signaling pathway was activated in response to radiation and that its activation by Wnt3a, a ligand-protein involved in the ß-catenin signaling pathway, inhibited apoptosis and the production of ROS in irradiated human keratinocyte HaCaT cells and skin fibroblast WS1 cells. Additionally, Wnt3a promoted cell migration after irradiation. In a mouse model of full-thickness skin wounds combined with total-body irradiation, Wnt3a was shown to facilitate skin wound healing. The results from RNA-Seq revealed that 24 genes were upregulated and 154 were downregulated in Wnt3a-treated irradiated skin cells, and these dysregulated genes were mainly enriched in the tight junction pathway. Among them, Marvel D3 showed the most obvious difference. We further found that the activated ß-catenin signaling pathway stimulated the phosphorylation of JNK by silencing Marvel D3. Treatment of irradiated cells with SP600125, a JNK inhibitor, augmented ROS production and impeded cell migration. Furthermore, treatment with Wnt3a or transfection with Marvel D3-specific siRNAs could reverse the above effects. Taken together, these findings illustrate that activated ß-catenin signaling stimulates the activation of JNK by negatively regulating Marvel D3 to ameliorate radiation-induced skin injury.

Design and synthesis of novel fluorescently labeled analogs of vemurafenib targeting MKK4.

The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent compound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suitable tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of MKK4.

JNK­IN­8 treatment alleviates lipopolysaccharide­induced acute lung injury via suppression of inflammation and oxidative stress regulated by JNK/NF­κB signaling.

JNK serves critical roles in numerous types of inflammation­ and oxidative stress­induced disease, including acute lung injury (ALI). JNK­IN­8 is the first irreversible JNK inhibitor that has been described. However, whether JNK­IN­8 can prevent lipopolysaccharide (LPS)­induced ALI by inhibiting JNK activation and its downstream signaling is poorly understood. The objective of the present study was to investigate the specific therapeutic effects of JNK­IN­8 on LPS­induced ALI and the molecular mechanisms involved. JNK­IN­8 attenuated myeloperoxidase activity, malondialdehyde and superoxide dismutase content and the lung wet/dry ratio, and improved the survival rate following lethal injection of LPS. Additionally, JNK­IN­8 decreased bronchoalveolar lavage fluid protein levels, lactate dehydrogenase activity, neutrophil infiltration and the number of macrophages (as demonstrated by flow cytometry), as well as the production of TNF­α, IL­6 and IL­1ß (as evaluated via ELISA). In addition, reverse transcription­quantitative PCR and ELISA showed that JNK­IN­8 attenuated LPS­induced inflammatory cytokine production and oxidative stress in primary murine peritoneal macrophages and RAW264.7 cells in vitro. Furthermore, the present study demonstrated that the JNK/NF­κB signaling pathway was involved in the therapeutic effect of JNK­IN­8 against LPS­induced injury both in vivo and in vitro. In conclusion, these findings indicated that JNK­IN­8 had a therapeutic effect on LPS­induced ALI in mice. The mechanism may be associated with inhibition of the JNK/NF­κB signaling pathway. JNK­IN­8 may be a potential therapeutic agent for the treatment of ALI.

From off-to on-target: New BRAF-inhibitor-template-derived compounds selectively targeting mitogen activated protein kinase kinase 4 (MKK4).

The mitogen-activated protein kinase (MAP) kinase 4 (MKK4) was found to be a major regulator of liver regeneration and could be a valuable drug target addressing liver related diseases by restoring its intrinsic regenerative capacity. We report on the synthesis and optimization of novel MKK4 inhibitors following a target-hopping strategy from the FDA-approved BRAFV600E inhibitor PLX4032 (8). Applying an iterative multi-parameter optimization process we carved out essential structural features yielding in compounds with a low nanomolar affinity for MKK4 and excellent selectivity profiles against the main off-targets MKK7 and JNK1, which, upon relevant inhibition, would totally abrogate the pro-regenerative effect of MKK4 inhibition, as well as against the off-targets MAP4K5, ZAK and BRAF with selectivity factors ranging from 40 to 430 for our best-balanced compounds 70 and 73.

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition.

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.

Inhibitory Effect of a Human MicroRNA, miR-6133-5p, on the Fibrotic Activity of Hepatic Stellate Cells in Culture.

BACKGROUND: We recently identified 39 human microRNAs, which effectively suppress hepatitis B virus (HBV) replication in hepatocytes. Chronic HBV infection often results in active, hepatitis-related liver fibrosis; hence, we assessed whether any of these microRNAs have anti-fibrotic potential and predicted that miR-6133-5p may target several fibrosis-related genes. METHODS: The hepatic stellate cell line LX-2 was transfected with an miR-6133-5p mimic and subsequently treated with Transforming growth factor (TGF)-ß. The mRNA and protein products of the COL1A1 gene, encoding collagen, and the ACTA2 gene, an activation marker of hepatic stellate cells, were quantified. RESULTS: The expression of COL1A1 and ACTA2 was markedly reduced in LX-2 cells treated with miR-6133-5p. Interestingly, phosphorylation of c-Jun N-terminal kinase (JNK) was also significantly decreased by miR-6133-5p treatment. The expression of several predicted target genes of miR-6133-5p, including TGFBR2 (which encodes Transforming Growth Factor Beta Receptor 2) and FGFR1 (which encodes Fibroblast Growth Factor Receptor 1), was also reduced in miR-6133-5p-treated cells. The knockdown of TGFBR2 by the corresponding small interfering RNA greatly suppressed the expression of COL1A1 and ACTA2. Treatment with the JNK inhibitor, SP600125, also suppressed COL1A1 and ACTA2 expression, indicating that TGFBR2 and JNK mediate the anti-fibrotic effect of miR-6133-5p. The downregulation of FGFR1 may result in a decrease of phosphorylated Akt, ERK (extracellular signal-regulated kinase), and JNK. CONCLUSION: miR-6133-5p has a strong anti-fibrotic effect, mediated by inactivation of TGFBR2, Akt, and JNK.

Discovery of Covalent MKK4/7 Dual Inhibitor.

MKK4/7 are kinases that phosphorylate JNKs and regulate the MAPK signaling pathway. Their overexpression has been associated with tumorigenesis and aggressiveness in cancers such as breast, prostate, non-small cell lung, and pediatric leukemia, making them a potential target for inhibitor development. Here, we report the discovery, development, and validation of a dual MKK4/7 inhibitor, BSJ-04-122, that covalently targets a conserved cysteine located before the DFG motif and displays excellent kinome selectivity. BSJ-04-122 exhibits potent cellular target engagement and induces robust target-specific downstream effects. The combination of the dual MKK4/7 inhibitor with a selective, covalent JNK inhibitor demonstrated an enhanced antiproliferative activity against triple-negative breast cancer cells. Taken together, the results show that BSJ-04-122 represents a pharmacological probe for MKK4/7 and credential covalent targeting as a way to explore the therapeutic potential of these kinases.

P38K and JNK pathways are induced by amyloid-ß in astrocyte: Implication of MAPK pathways in astrogliosis in Alzheimer's disease.

Astrocyte activation is one of the crucial hallmarks of Alzheimer's disease (AD) along with amyloid-ß (Aß) plaques, neurofibrillary tangles and neuron death. Glial scar and factors secreted from activated astrocytes have important contribution on neuronal health in AD. In this study, we investigated the mechanisms of astrocyte activation both in in vitro and in vivo models of AD. In this regard, mitogen activated protein kinase (MAPK) signalling cascades that control several fundamental and stress related cellular events, has been implicated in astrocyte activation in various neurological diseases. We checked activation of different MAPKs by western blot and immunocytochemistry and found that both JNK and p38K, but not ERK pathways are activated in Aß-treated astrocytes in culture and in Aß-infused rat brain cortex. Next, to investigate the downstream consequences of these two MAPKs (JNK and p38K) in Aß-induced astrocyte activation, we individually blocked these pathways by specific inhibitors in presence and absence of Aß and checked Aß-induced cellular proliferation, morphological changes and glial fibrillary acidic protein (GFAP) upregulation. We found that activation of both JNK and p38K signalling cascades are involved in astrocyte proliferation evoked by Aß, whereas only p38K pathway is implicated in morphological changes and GFAP upregulation in astrocytes exposed to Aß. To further validate the implication of p38K pathway in Aß-induced astrocyte activation, we also observed that transcription factor ATF2, a downstream phosphorylation substrate of p38, is phosphorylated upon Aß treatment. Taken together, our study indicates that p38K and JNK pathways mediate astrocyte activation and both the pathways are involved in cellular proliferation but only p38K pathway contributes in morphological changes triggered by Aß.