The MKK3 MAPK cascade integrates temperature and after-ripening signals to modulate seed germination.

The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.

MKK3 depletion attenuates intestinal injury after traumatic hemorrhagic shock by restoring mitochondrial function.

BACKGROUND: Traumatic hemorrhagic shock (THS) is a complex pathophysiological process resulting in multiple organ failure. Intestinal barrier dysfunction is one of the mechanisms implicated in multiple organ failure. The present study aimed to explore the regulatory role of mitogen-activated protein kinase kinase 3 (MKK3) in THS-induced intestinal injury and to elucidate its potential mechanism. METHODS: Rats were subjected to trauma and hemorrhage to establish a THS animal model. MKK3-targeted lentiviral vectors were injected via the tail vein 72 h before modeling. Twelve hours post-modeling, the mean arterial pressure (MAP) and heart rate (HR) were monitored, and histological injury to the intestine was assessed via H&E staining and transmission electron microscopy. Mitochondrial function and mitochondrial reactive oxygen species (ROS) were evaluated. IEC-6 cells were exposed to hypoxia to mimic intestinal injury following THS in vitro. RESULTS: MKK3 deficiency alleviated intestinal injury and restored mitochondrial function in intestinal tissues from THS-induced rats and hypoxia-treated IEC-6 cells. In addition, MKK3 deficiency promoted Sirt1/PGC-1α-mediated mitochondrial biogenesis and restricted Pink1/Parkin-mediated mitophagy in the injured intestine and IEC-6 cells. Furthermore, the protective effect of MKK3 knockdown against hypoxia-induced mitochondrial damage was strengthened upon simultaneous LC3B/Pink1/Parkin knockdown or weakened upon simultaneous Sirt1 knockdown. CONCLUSION: MKK3 deficiency protected against intestinal injury induced by THS by promoting mitochondrial biogenesis and restricting excessive mitophagy.

Discovery of pyrazolo[3,4-d]pyrimidines as novel mitogen-activated protein kinase kinase 3 (MKK3) inhibitors.

The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure-activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3's role in various cancers.

Activated MKK3/MYC crosstalk impairs dabrafenib response in BRAFV600E colorectal cancer leading to resistance.

Colorectal cancer (CRC) patients with BRAF mutations develop resistance to BRAF inhibitors at a very early stage. Understanding the molecular mechanisms involved in BRAF inhibitor resistance is critical for the development of novel therapeutic opportunities for this subtype of CRC patients. CRC cells bearing BRAF mutations are mostly sensitive to the abrogation of Mitogen-Activated Protein Kinase Kinase 3 (MKK3), a specific activator of p38MAPKs signaling, suggesting that BRAF alterations might addict CRC cells to the MKK3/p38MAPK signaling. Interestingly, publicly available gene expression profiling data show significantly higher MKK3 transcript levels in CRC lines with acquired resistance to BRAF inhibitors. Herein, we investigated the roles of MKK3 in the response to BRAF targeting (dabrafenib) with COLO205 and HT29 BRAFV600E CRC lines and derived dabrafenib-resistant (DABR) sublines. Dabrafenib treatments reduce MKK3 activation by inducing autophagy in parental but not DABR cells. The MKK3 knockdown induces cell death in DABR cells, whereas ectopic MKK3 expression reduces dabrafenib sensitivity in parental cells. Mechanistically, activated MKK3 interacts and co-localizes with c-Myc oncoprotein (MYC), sustaining MYC protein stability and thus preventing the dabrafenib induced effects in CRC DABR cells both in vitro and in vivo. Overall, we identify a novel molecular mechanism beyond the dabrafenib resistance, shedding light on an uncovered vulnerability for the development of novel therapeutic opportunities in BRAFV600E CRC.

Identification of MAP Kinase Kinase 3 as a protein target of myricetin in non-small cell lung cancer cells.

Myricetin is a typical flavonol with various pharmacological effects which shows favorable biological activities in cancer. However, the underlying mechanisms and potential targets of myricetin in NSCLC (non-small cell lung cancer) cells remain unclear. First, we demonstrated that myricetin not only inhibited the proliferation, migration and invasion, but also induced apoptosis in A549 and H1299 cells in a dose-dependent manner. Then, we confirmed myricetin may play an anti-NSCLC effect through modulating MAPK-related functions and signaling pathway by Network pharmacology. Furthermore, MKK3 (MAP Kinase Kinase 3) was identified and confirmed as a potential target of myricetin by biolayer interferometry (BLI) and molecular docking, revealing that myricetin directly bound to MKK3. Moreover, three mutations (D208, L240, and Y245) of key amino acids predicted by molecular docking obviously decreased the affinity between myricetin and MKK3. Finally, enzyme activity assay was utilized to determine the effect of myricetin on MKK3 activity in vitro, and the result showed that myricetin attenuated MKK3 activity. Subsequently, myricetin decreased the phosphorylation of p38 MAPK. Furthermore, knockdown of MKK3 reduced the susceptibility of A549 and H1299 cells to myricetin. These results suggested that myricetin inhibited the growth of NSCLC cells via targeting MKK3 and influencing the downstream p38 MAPK signaling pathway. The findings revealed that MKK3 is a potential target of myricetin in the NSCLC and myricetin is considered to be a small-molecular inhibitor of MKK3, which can improve comprehension of the molecular mechanisms of myricetin pharmacological effects in cancer and further development of MKK3 inhibitors.

ENPP1 deletion causes mouse osteoporosis via the MKK3/p38 MAPK/PCNA signaling pathway.

BACKGROUND: Apart from the current understanding of enzyme function, the mechanism of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) deficiency-associated osteoporosis is unknown. We aimed to explore the changes in the expression of signaling pathways of bone tissues involved in Enpp1 deficiency. METHODS: The body weights and morphology and histology of the bones of male Enpp1 knockout (KO) and wild-type (WT) mice were assessed. The humeri of WT and Enpp1 KO mice at 12 weeks of age were subjected to high-throughput quantitative molecular measurements, and bioinformatics analysis was performed. Proteins from humeri and calvarial pre-osteoblasts (Pobs) were used to verify the differentially expressed signaling pathways and to explain the mechanism of Enpp1 deficiency-associated osteoporosis. RESULTS: Enpp1 KO mice had significantly lower body weight and trabecular bone mass in the hindlimbs than WT mice. Proteomics and immunoblotting showed that Enpp1 deletion downregulated the expression of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in bones. Lysophosphatidic acid (LPA) was involved in activating the MKK3/p38 MAPK/PCNA pathway and proliferating Pobs in Enpp1 KO mice, whereas a p38 MAPK inhibitor suppressed the LPA-induced pro-proliferation phenotype (p < 0.05). CONCLUSION: The inhibition of MKK3/p38 MAPK/PCNA pathway plays an important role in the development of osteoporosis caused by Enpp1 deficiency, and LPA partially rescued the proliferation of pre-osteoblasts via the MKK3/p38 MAPK/PCNA pathway.

Regulation of the p38-MAPK pathway by hyperosmolarity and by WNK kinases.

p38-MAPK is a stress-response kinase activated by hyperosmolarity. Here we interrogated the pathways involved. We show that p38-MAPK signaling is activated by hyperosmotic stimulation in various solutions, cell types and colonic organoids. Hyperosmolarity sensing is detected at the level of the upstream activators of p38-MAPK: TRAF2/ASK1 (but not Rac1) and MKK3/6/4. While WNK kinases are known osmo-sensors, we found, unexpectedly, that short (2 h) inhibition of WNKs (with WNK463) led to elevated p38-MAPK activity under hyperosmolarity, which was mediated by WNK463-dependent stimulation of TAK1 or TRAF2/ASK1, the upstream activators of MKK3/6/4. However, this effect was temporary and was reversed by long-term (2 days) incubation with WNK463. Accordingly, 2 days (but not 2 h) inhibition of p38-MAPK or its upstream activators ASK1 or TAK1, or WNKs, diminished regulatory volume increase (RVI) following cell shrinkage under hyperosmolarity. We also show that RVI mediated by the ion transporter NKCC1 is dependent on p38-MAPK. Since WNKs are known activators of NKCC1, we propose a WNK- > NKCC1- > p38-MAPK pathway that controls RVI. This pathway is augmented by NHE1. Additionally, hyperosmolarity inhibited mTORC1 activation and cell proliferation. Thus, activation of p38-MAPK and WNKs is important for RVI and for cell proliferation.

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38ß, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.

The human heart can increase its size to supply more blood to the body's organs. This process, called hypertrophy, can happen during exercise or be caused by medical conditions, such as high blood pressure or inherited genetic diseases. If hypertrophy is continually driven by illness, this can cause the heart to fail and no longer be able to properly pump blood around the body. For hypertrophy to happen, several molecular changes occur in the cells responsible for contracting the heart, including activation of the p38 pathway. Within this pathway is a p38 enzyme as well as a series of other proteins which are sequentially turned on in response to stress, such as inflammatory molecules or mechanical forces that alter the cell's shape. There are different types of p38 enzyme which have been linked to other diseases, making them a promising target for drug development. However, clinical trials blocking individual members of the p38 family have had disappointing results. An alternative approach is to target other proteins involved in the p38 pathway, such as MKK6, but it is not known what effect this might have. To investigate, Romero-Becerra et al. genetically modified mice to not have any MKK6 protein. As a result, these mice had a shorter lifespan, with hypertrophy developing at a young age that led to heart problems. Romero-Becerra et al. used different mice models to understand why this happened, showing that a lack of MKK6 reduces the activity of a specific member of the p38 family called p38α. However, this blockage boosted a different branch of the pathway which involved two other p38 proteins, p38γ and p38δ. This, in turn, triggered another key pathway called mTOR which also promotes hypertrophy of the heart. These results suggest that drugs blocking MKK6 and p38α could lead to side effects that cause further harm to the heart. A more promising approach for treating hypertrophic heart conditions could be to inhibit p38γ and/or p38δ. However, before this can be fully explored, further work is needed to generate compounds that specifically target these proteins.

Gamma synuclein promotes cancer metastasis through the MKK3/6-p38MAPK cascade.

Gamma synuclein (SNCG) is a neuronal protein that is also aberrantly overexpressed in various types of human cancer. SNCG overexpression promotes cancer invasion and metastasis. However, the mechanisms that drive cancer metastasis upon SNCG expression remain elusive. Elucidation of the mechanisms underlying the promotion of cancer metastasis by SNCG may help discover therapeutic avenues for SNCG-overexpressed cancer. Here, we show that SNCG promotes transforming growth factor-ß (TGF-ß)-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. Mechanistically, SNCG promotes p38MAPK phosphorylation by interacting with the MAPK kinase 3/6 (MKK3/6) and prevents their degradation. SNCG knockdown leads to a decrease in TGF-ß-induced phosphorylation of MKK3/6; and abrogates the induction of matrix metalloproteinase (MMP)-9 expression by TGF-ß and its target gene Twist1. Furthermore, p38MAPK inhibition abrogates the promotion of MMP-9 expression and cancer cell invasion by SNCG. Both p38MAPK and MMP inhibitors can suppress the promotion of cancer cell invasion by SNCG. Finally, overexpression of SNCG in liver cancer cells promotes lung metastasis, which can be suppressed by the p38MAPK inhibitor. Together, our data uncover a previously unknown role of SNCG in promoting TGF-ß-MKK3/6-p38MAPK signaling. This study highlights the critical role of p38MAPK in the promotion of cancer metastasis by SNCG, and indicates that p38MAPK inhibitor may serve as a potential therapeutic for SNCG-overexpressed cancer.

Fenbendazole and its synthetic analog interfere with HeLa cells' proliferation and energy metabolism via inducing oxidative stress and modulating MEK3/6-p38-MAPK pathway.

Fenbendazole, a broad-spectrum anti-parasitic drug, can be a potential anti-tumor agent. In this study, we synthesized and purified its derivative, analog 6, intending to achieve improved efficacy in cancer cells and decreased toxicity in normal cells. To evaluate in vitro anti-tumor activities of fenbendazole and analog 6 in different cancer cell lines, a CCK-8 assay was performed, and we found that human cervical cancer HeLa cells were more sensitive to analog 6 than to fenbendazole. Furthermore, we explored the associated mechanism, and our results showed that analog 6 and fenbendazole could induce oxidative stress by accumulating ROS. It not only activated the p38-MAPK signaling pathway, thereby inhibiting the proliferation of HeLa cells and enhancing the apoptosis of HeLa cells, but also significantly induced impaired energy metabolism and restrained their migration and invasion. In addition, the modified analog 6 showed reduced toxicity to normal cells without decreased anti-cancer effect. In conclusion, fenbendazole and analog 6 have multiple targets and strong anti-tumor effects on HeLa cells in vitro and in vivo. The optimized analog 6 could inhibit the viability of HeLa cells with lower toxicity than normal human cells, promising to be developed as an antitumor active compound.

Discovery of First-in-Class TAK1-MKK3 Protein-Protein Interaction (PPI) Inhibitor (R)-STU104 for the Treatment of Ulcerative Colitis through Modulating TNF-α Production.

Tumor necrosis factor α (TNF-α) has been demonstrated to be a therapeutic target for autoimmune diseases. However, this biological therapy exhibits some inevitable disadvantages, such as risk of infection. Thus, small-molecule alternatives by targeting TNF-α production signaling pathway are still in demand. Herein, we describe the design, synthesis, and structure-activity relationships of 3-aryindanone compounds regarding their modulation of TNF-α production. Among them, (R)-STU104 exhibited the most potent inhibitory activity on TNF-α production, which suppressed the TAK1/MKK3/p38/MnK1/MK2/elF4E signal pathways through binding with MKK3 and disrupting the TAK1 phosphorylating MKK3. As a result, (R)-STU104 demonstrated remarkable dose-effect relationships on both acute and chronic mouse UC models. In addition to its good pharmacokinetic (PK) and safety profile, (R)-STU104 showed better anti-UC efficacy in vivo at 10 mg/kg/d than mesalazine at the dose of 50 mg/kg/d. These results suggested that TAK1-MKK3 interaction inhibitors could be potentially utilized for the treatment of UC.

Receptor for advanced glycation end products aggravates cognitive deficits in type 2 diabetes through binding of C-terminal AAs 2-5 to mitogen-activated protein kinase kinase 3 (MKK3) and facilitation of MEKK3-MKK3-p38 module assembly.

In this study, we explored the precise mechanisms underlying the receptor for advanced glycation end products (RAGE)-mediated neuronal loss and behavioral dysfunction induced by hyperglycemia. We used immunoprecipitation (IP) and GST pull-down assays to assess the interaction between RAGE and mitogen-activated protein kinase kinase 3 (MKK3). Then, we investigated the effect of specific mutation of RAGE on plasticity at hippocampal synapses and behavioral deficits in db/db mice through electrophysiological recordings, morphological assays, and behavioral tests. We discovered that RAGE binds MKK3 and that this binding is required for assembly of the MEKK3-MKK3-p38 signaling module. Mechanistically, we found that activation of p38 mitogen-activated protein kinase (MAPK)/NF-κB signaling depends on mediation of the RAGE-MKK3 interaction by C-terminal RAGE (ctRAGE) amino acids (AAs) 2-5. We found that ctRAGE R2A-K3A-R4A-Q5A mutation suppressed neuronal damage, improved synaptic plasticity, and alleviated behavioral deficits in diabetic mice by disrupting the RAGE-MKK3 conjugation. High glucose induces direct binding of RAGE and MKK3 via ctRAGE AAs 2-5, which leads to assembly of the MEKK3-MKK3-p38 signaling module and subsequent activation of the p38MAPK/NF-κB pathway, and ultimately results in diabetic encephalopathy (DE).

QTL x environment modeling of malting barley preharvest sprouting.

KEY MESSAGE: HvMKK3 alleles are temperature sensitive and are major contributors to environmental stability of preharvest sprouting in barley. Preharvest sprouting (PHS) can severely damage barley (Hordeum vulgare L.) malting quality, but PHS resistance is often negatively correlated with malting quality. Seed dormancy is closely related to PHS. Increased temperature during grain fill can decrease seed dormancy in barley, but genetic components of seed dormancy temperature sensitivity are poorly understood. Six years of PHS data were used to fit quantitative trait locus (QTL) x environment mixed models incorporating marker data from seed dormancy genes HvAlaAT1, HvGA20ox1, and HvMKK3 and weather covariates in spring and winter two-row malting barley. Variation in winter barley PHS was best modeled by average temperature range during grain fill and spring barley PHS by total precipitation during grain fill. Average high temperature during grain fill also accurately modeled PHS for both datasets. A highly non-dormant HvMKK3 allele determined baseline PHS susceptibility and HvAlaAT1 interactions with multiple HvMKK3 alleles conferred environmental sensitivity. Polygenic variation for PHS within haplotype was detected. Residual genotype and QTL by environment interaction variance indicated additional environmental and genetic factors involved in PHS. These models provide insight into genotype and environmental regulation of barley seed dormancy, a method for PHS forecasting, and a tool for breeders to improve PHS resistance.

Quadruple and Truncated MEK3 Mutants Identified from Acute Lymphoblastic Leukemia Promote Degradation and Enhance Proliferation.

Compared to other ethnicities, Hispanic children incur the highest rates of leukemia, and most cases are diagnosed as Acute Lymphoblastic Leukemia (ALL). Despite improved treatment and survival for ALL, disproportionate health outcomes in Hispanics persist. Thus, it is essential to identify oncogenic mutations within this demographic to aid in the development of new strategies to diagnose and treat ALL. Using whole-exome sequencing, five single nucleotide polymorphisms within mitogen-activated protein kinase 3 (MAP2K3) were identified in an ALL cancer patient library from the U.S./Mexico border. MAP2K3 R26T and P11T are located near the substrate-binding site, while R65L and R67W localized to the kinase domain. Truncated-MAP2K3 mutant Q73* was also identified. Transfection in HEK293 cells showed that the quadruple-MEK3 mutant (4M-MEK3) impacted protein stability, inducing degradation and reducing expression. The expression of 4M-MEK3 could be rescued by cysteine/serine protease inhibition, and proteasomal degradation of truncated-MEK3 occurred in a ubiquitin-independent manner. MEK3 mutants displayed reduced auto-phosphorylation and enzymatic activity, as seen by decreases in p38 phosphorylation. Furthermore, uncoupling of the MEK3/p38 signaling pathway resulted in less suppressive activity on HEK293 cell viability. Thus, disruption of MEK3 activation may promote proliferative signals in ALL. These findings suggest that MEK3 represents a potential therapeutic target for treating ALL.

Myostatin Deficiency Protects C2C12 Cells from Oxidative Stress by Inhibiting Intrinsic Activation of Apoptosis.

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn-/- cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn-/- cells and could serve as basis for further research.

Discovery of the first chemical tools to regulate MKK3-mediated MYC activation in cancer.

The transcription master regulator MYC plays an essential role in regulating major cellular programs and is a well-established therapeutic target in cancer. However, MYC targeting for drug discovery is challenging. New therapeutic approaches to control MYC-dependent malignancy are urgently needed. The mitogen-activated protein kinase kinase 3 (MKK3) binds and activates MYC in different cell types, and disruption of MKK3-MYC protein-protein interaction may provide a new strategy to target MYC-driven programs. However, there is no perturbagen available to interrogate and control this signaling arm. In this study, we assessed the drugability of the MKK3-MYC complex and discovered the first chemical tool to regulate MKK3-mediated MYC activation. We have designed a short 44-residue inhibitory peptide and developed a cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay to discover the first small molecule MKK3-MYC PPI inhibitor. We have optimized and miniaturized the assay into an ultra-high-throughput screening (uHTS) 1536-well plate format. The pilot screen of ~6,000 compounds of a bioactive chemical library followed by multiple secondary and orthogonal assays revealed a quinoline derivative SGI-1027 as a potent inhibitor of MKK3-MYC PPI. We have shown that SGI-1027 disrupts the MKK3-MYC complex in cells and in vitro and inhibits MYC transcriptional activity in colon and breast cancer cells. In contrast, SGI-1027 does not inhibit MKK3 kinase activity and does not interfere with well-known MKK3-p38 and MYC-MAX complexes. Together, our studies demonstrate the drugability of MKK3-MYC PPI, provide the first chemical tool to interrogate its biological functions, and establish a new uHTS assay to enable future discovery of potent and selective inhibitors to regulate this oncogenic complex.

Knockdown of Mitogen-Activated Protein Kinase Kinase 3 Negatively Regulates Hepatitis A Virus Replication.

Zinc chloride is known to be effective in combatting hepatitis A virus (HAV) infection, and zinc ions seem to be especially involved in Toll-like receptor (TLR) signaling pathways. In the present study, we examined this involvement in human hepatoma cell lines using a human TLR signaling target RT-PCR array. We also observed that zinc chloride inhibited mitogen-activated protein kinase kinase 3 (MAP2K3) expression, which could downregulate HAV replication in human hepatocytes. It is possible that zinc chloride may inhibit HAV replication in association with its inhibition of MAP2K3. In that regard, this study set out to determine whether MAP2K3 could be considered a modulating factor in the development of the HAV pathogen-associated molecular pattern (PAMP) and its triggering of interferon-ß production. Because MAP2K3 seems to play a role in antiviral immunity against HAV infection, it is a promising target for drug development. The inhibition of MAP2K3 may also prevent HAV patients from developing a severe hepatitis A infection.

MTA2 silencing attenuates the metastatic potential of cervical cancer cells by inhibiting AP1-mediated MMP12 expression via the ASK1/MEK3/p38/YB1 axis.

Metastasis-associated protein 2 (MTA2) is a transcription factor that is highly associated with matrix metalloproteinase 12 (MMP12). Thus, we hypothesized that MTA2 may regulate MMP12 expression and is involved in cervical cancer metastasis. Results showed that MTA2 and MMP12 were highly expressed in cervical cancer cells, and MTA2 knockdown reduced MMP12 expression and inhibited the metastasis of cervical cancer cells in xenograft mice. MMP12 knockdown did not influence the viability of cervical cancer cells but clearly inhibited cell migration and invasion both in vitro and in vivo. MMP12 was highly expressed in cervical tumor tissues and correlated with the poor survival rate of patients with cervical cancer. Further investigations revealed that p38 mitogen-activated protein kinase (p38), mitogen-activated protein kinase kinase 3 (MEK3), and apoptosis signal-regulating kinase 1 (ASK1) were involved in MMP12 downregulation in response to MTA2 knockdown. Results also demonstrated that p38-mediated Y-box binding protein1 (YB1) phosphorylation disrupted the binding of AP1 (c-Fos/c-Jun) to the MMP12 promoter, thereby inhibiting MMP12 expression and the metastatic potential of cervical cancer cells. Collectively, targeting both MTA2 and MMP12 may be a promising strategy for the treatment of cervical cancer.

Cutaneous innate immune tolerance is mediated by epigenetic control of MAP2K3 by HDAC8/9.

The skin typically tolerates exposure to various microbes and chemicals in the environment. Here, we investigated how the epidermis maintains this innate immune tolerance to stimuli that are recognized by Toll-like receptors (TLRs). Loss of tolerance to TLR ligands occurred after silencing of the histone deacetylases (HDACs) HDAC8 and HDAC9 in keratinocytes. Transcriptional analysis identified MAP2K3 as suppressed by HDAC8/9 activity and a potential key intermediary for establishing this tolerance. HDAC8/9 influenced acetylation at H3K9 and H3K27 marks in the MAP2K3 promoter. Proteomic analysis further identified SSRP1 and SUPT16H as associated with HDAC8/9 and responsible for transcriptional elongation of MAP2K3. Silencing of MAP2K3 blocked the capacity of HDAC8/9 to influence cytokine responses. Relevance in vivo was supported by observations of increased MAP2K3 in human inflammatory skin conditions and the capacity of keratinocyte HDAC8/9 to influence dendritic cell maturation and T cell proliferation. Keratinocyte-specific deletion of HDAC8/9 also increased inflammation in mice after exposure to ultraviolet radiation, imiquimod, or Staphylococcus aureus These findings define a mechanism for the epidermis to regulate inflammation in the presence of ubiquitous TLR ligands.