Phosphorylation Of HDAC4 Research Articles

Nesfatin-1 enhances vascular smooth muscle calcification through facilitating BMP-2 osteogenic signaling

Vascular calcification (VC) arises from the accumulation of calcium salts in the intimal or tunica media layer of the aorta, contributing to higher risk of cardiovascular events and mortality. Despite this, the mechanisms driving VC remain incompletely understood. We previously described that nesfatin-1 functioned as a switch for vascular smooth muscle cells (VSMCs) plasticity in hypertension and neointimal hyperplasia. In this study, we sought to investigate the role and mechanism of nesfatin-1 in VC. The expression of nesfatin-1 was measured in calcified VSMCs and aortas, as well as in patients. Loss- and gain-of-function experiments were evaluated the roles of nesfatin-1 in VC pathogenesis. The transcription activation of nesfatin-1 was detected using a mass spectrometry. We found higher levels of nesfatin-1 in both calcified VSMCs and aortas, as well as in patients with coronary calcification. Loss-of-function and gain-of-function experiments revealed that nesfatin-1 was a key regulator of VC by facilitating the osteogenic transformation of VSMCs. Mechanistically, nesfatin-1 promoted the de-ubiquitination and stability of BMP-2 via inhibiting the E3 ligase SYTL4, and the interaction of nesfatin-1 with BMP-2 potentiated BMP-2 signaling and induced phosphorylation of Smad, followed by HDAC4 phosphorylation and nuclear exclusion. The dissociation of HDAC4 from RUNX2 elicited RUNX2 acetylation and subsequent nuclear translocation, leading to the transcription upregulation of OPN, a critical player in VC. From a small library of natural compounds, we identified that Curculigoside and Chebulagic acid reduced VC development via binding to and inhibiting nesfatin-1. Eventually, we designed a mass spectrometry-based DNA-protein interaction screening to identify that STAT3 mediated the transcription activation of nesfatin-1 in the context of VC. Overall, our study demonstrates that nesfatin-1 enhances BMP-2 signaling by inhibiting the E3 ligase SYTL4, thereby stabilizing BMP-2 and facilitating the downstream phosphorylation of SMAD1/5/9 and HDAC4. This signaling cascade leads to RUNX2 activation and the transcriptional upregulation of MSX2, driving VC. These insights position nesfatin-1 as a potential therapeutic target for preventing or treating VC, advancing our understanding of the molecular mechanisms underlying this critical cardiovascular condition.

Genome-wide study reveals novel roles for formin-2 in axon regeneration as a microtubule dynamics regulator and therapeutic target for nerve repair

Peripheral nerves regenerate successfully; however, clinical outcome after injury is poor. We demonstrated that low-dose ionizing radiation (LDIR) promoted axon regeneration and function recovery after peripheral nerve injury (PNI). Genome-wide CpG methylation profiling identified LDIR-induced hypermethylation of the Fmn2 promoter, exhibiting injury-induced Fmn2 downregulation in dorsal root ganglia (DRGs). Constitutive knockout or neuronal Fmn2 knockdown accelerated nerve repair and function recovery. Mechanistically, increased microtubule dynamics at growth cones was observed in time-lapse imaging of Fmn2-deficient DRG neurons. Increased HDAC5 phosphorylation and rapid tubulin deacetylation were found in regenerating axons of neuronal Fmn2-knockdown mice after injury. Growth-promoting effect of neuronal Fmn2 knockdown was eliminated by pharmaceutical blockade of HDAC5 or neuronal Hdac5 knockdown, suggesting that Fmn2deletion promotes axon regeneration via microtubule post-translational modification. In silico screening of FDA-approved drugs identified metaxalone, administered either immediately or 24-h post-injury, accelerating function recovery. This work uncovers a novel axon regeneration function of Fmn2 and a small-molecule strategy for PNI.

Gαq-PKD/PKCμ signal regulating the nuclear export of HDAC5 to induce the IκB expression and limit the NF-κB-mediated inflammatory response essential for early pregnancy.

Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here, we demonstrated that knockout of Gαq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gαq resulted in over-activation of nuclear factor (NF)-κB signaling, due to the decreased expression of NFκBIA, which encode the IκB protein and is the negative regulator for NF-κB. Mechanistically, Gαq deficiency decreased the Protein kinase D (PKD, also called PKCμ) phosphorylation levels, leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HDAC5 retarded histone acetylation to inhibit the induced NFκBIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKCμ or inhibition of the HDAC5 restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gαq-deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gαq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gαq as a key regulator of the inflammatory cytokine's expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.

Type 2 diabetes susceptibility gene GRK5 regulates physiological pancreatic β-cell proliferation via phosphorylation of HDAC5

Restoring functional β cell mass is a potential therapy for those with diabetes. However, the pathways regulating β cell mass are not fully understood. Previously, we demonstrated that Sox4 is required for β cell proliferation during prediabetes. Here, we report that Sox4 regulates β cell mass through modulating expression of the type 2 diabetes (T2D) susceptibility gene GRK5. β cell-specific Grk5 knockout mice showed impaired glucose tolerance with reduced β cell mass, which was accompanied by upregulation of cell cycle inhibitor gene Cdkn1a. Furthermore, we found that Grk5 may drive β cell proliferation through a pathway that includes phosphorylation of HDAC5 and subsequent transcription of immediate-early genes (IEGs) such as Nr4a1, Fosb, Junb, Arc, Egr1, and Srf. Together, these studies suggest GRK5 is linked to T2D through regulation of β cell growth and that it may be a target to preserve β cells during the development of T2D.

Statins inhibit protein kinase D (PKD) activation in intestinal cells and prevent PKD1-induced growth of murine enteroids.

We examined the impact of statins on protein kinase D (PKD) activation by G protein-coupled receptor (GPCR) agonists. Treatment of intestinal IEC-18 cells with cerivastatin inhibited PKD autophosphorylation at Ser916 induced by angiotensin II (ANG II) or vasopressin in a dose-dependent manner with half-maximal inhibition at 0.2 µM. Cerivastatin treatment inhibited PKD activation stimulated by these agonists for different times (5-60 min) and blunted HDAC5 phosphorylation, a substrate of PKD. Other lipophilic statins, including simvastatin, atorvastatin, and fluvastatin also prevented PKD activation in a dose-dependent manner. Using IEC-18 cell lines expressing PKD1 tagged with EGFP (enhanced green fluorescent protein), cerivastatin or simvastatin blocked GPCR-mediated PKD1-EGFP translocation to the plasma membrane and its subsequent nuclear accumulation. Similar results were obtained in IEC-18 cells expressing PKD3-EGFP. Mechanistically, statins inhibited agonist-dependent PKD activation rather than acting directly on PKD catalytic activity since exposure to cerivastatin or simvastatin did not impair PKD autophosphorylation or PKD1-EGFP membrane translocation in response to phorbol dibutyrate, which bypasses GPCRs and directly stimulates PKC and PKD. Furthermore, cerivastatin did not inhibit recombinant PKD activity determined via an in vitro kinase assay. Using enteroids generated from intestinal crypt-derived epithelial cells from PKD1 transgenic mice as a model of intestinal regeneration, we show that statins oppose PKD1-mediated increase in enteroid area, complexity (number of crypt-like buds), and DNA synthesis. Our results revealed a previously unappreciated inhibitory effect of statins on receptor-mediated PKD activation and in opposing the growth-promoting effects of PKD1 on intestinal epithelial cells.

Small-Molecule Inhibition of MuRF1 Prevents Early Disuse-Induced Diaphragmatic Dysfunction and Atrophy.

In clinical conditions such as diaphragm paralysis or mechanical ventilation, disuse-induced diaphragmatic dysfunction (DIDD) is a condition that poses a threat to life. MuRF1 is a key E3-ligase involved in regulating skeletal muscle mass, function, and metabolism, which contributes to the onset of DIDD. We investigated if the small-molecule mediated inhibition of MuRF1 activity (MyoMed-205) protects against early DIDD after 12 h of unilateral diaphragm denervation. Wistar rats were used in this study to determine the compound's acute toxicity and optimal dosage. For potential DIDD treatment efficacy, diaphragm contractile function and fiber cross-sectional area (CSA) were evaluated. Western blotting investigated potential mechanisms underlying MyoMed-205's effects in early DIDD. Our results indicate 50 mg/kg bw MyoMed-205 as a suitable dosage to prevent early diaphragmatic contractile dysfunction and atrophy following 12 h of denervation without detectable signs of acute toxicity. Mechanistically, treatment did not affect disuse-induced oxidative stress (4-HNE) increase, whereas phosphorylation of (ser632) HDAC4 was normalized. MyoMed-205 also mitigated FoxO1 activation, inhibited MuRF2, and increased phospho (ser473) Akt protein levels. These findings may suggest that MuRF1 activity significantly contributes to early DIDD pathophysiology. Novel strategies targeting MuRF1 (e.g., MyoMed-205) have potential therapeutic applications for treating early DIDD.

A signalling pathway for transcriptional regulation of sleep amount in mice.

In mice and humans, sleep quantity is governed by genetic factors and exhibits age-dependent variation1-3. However, the core molecular pathways and effector mechanisms that regulate sleep duration in mammals remain unclear. Here, we characterize a major signalling pathway for the transcriptional regulation of sleep in mice using adeno-associated virus-mediated somatic genetics analysis4. Chimeric knockout of LKB1 kinase-an activator of AMPK-related protein kinase SIK35-7-in adult mouse brain markedly reduces the amountand delta power-a measure of sleep depth-of non-rapid eye movement sleep (NREMS). Downstream of the LKB1-SIK3 pathway, gain or loss-of-function of the histone deacetylases HDAC4 and HDAC5 in adult brain neurons causes bidirectional changes of NREMS amount and delta power. Moreover, phosphorylation of HDAC4 and HDAC5 is associated with increasedsleep need, and HDAC4 specifically regulates NREMS amount in posterior hypothalamus. Genetic and transcriptomic studies reveal that HDAC4 cooperates with CREB in both transcriptional and sleep regulation. These findings introduce the concept of signalling pathways targeting transcription modulators to regulate daily sleep amount and demonstrate the power of somatic genetics in mouse sleep research.

Abstract 10039: Cardiomyocyte N-myristoylation Plays an Essential Role During the Development of Heart Failure

Background: N-myristoylation is co- and post-translational modifications of proteins, which involve attachment of myristic acid to the N-terminal glycine. Although N-myristoylation plays important roles in physiological activities such as intracellular signaling, the precise regulation and functional relevance of N-myristoylation in the heart remain to be elucidated. Methods and Results: We applied a click chemistry-based approach to determine the global N-myristoylated protein profiling in the cardiomyocyte. Proteomics analysis identified 41 and 34 N-myristoylated proteins in H9c2 myocytes and neonatal rat cardiomyocytes, and 66.1% of them were discovered for the first time at endogenous levels. The levels of N-myristoylation were significantly decreased by the N-myristoyltransferase (NMT) 2 inhibition with siRNA or adenoviral shRNA knockdown, but not by the NMT1 inhibition. In response to angiotensin II (AII), N-myristoylated levels were differentially altered, and MARCKS was identified as the most down-regulated N-myristoylated protein. Wild-type MARCKS was localized to the cellular membrane, while the mutant MARKS with replacement of N-terminal glycine to alanine (G2A), which lacked the capacity of N-myristoylation, exhibited cytosolic distribution. Cardiomyocyte hypertrophy was exacerbated in mutant G2A MARCKS accompanied by increased levels of histone acetylation and phosphorylation of HDAC4 and CaMKII after AII. Gene transfer delivering NMT2 gene specific to the heart using adeno-associated virus 9 significantly improved pressure overload-induced left ventricular systolic dysfunction, cardiac hypertrophy, and myocardial fibrosis, associated with decreased levels of histone acetylation after the transverse aortic constriction in mice ( Fig ). Conclusion: Cardiomyocyte N-myristoylation plays an essential role in developing heart failure, and its modification by NMT2 may be a novel therapeutic strategy for heart failure.

Angiotensin II Mediates Cardiomyocyte Hypertrophy in Atrial Cardiomyopathy via Epigenetic Transcriptional Regulation.

Aims European Heart Rhythm Association established an expert consensus to define, characterize, and classify atrial cardiomyopathy into four subgroups based on their histopathological features. The predominant pathological feature of classes I and III is the hypertrophy of atrial cardiomyocytes. Here, we aim to investigate the mechanism of epigenetic transcriptional regulation of cardiomyocyte hypertrophy in atrial cardiomyopathy. Methods and Results Compared with that of sinus rhythm control individuals, the myocardium of patients with atrial fibrillation exhibited increased levels of angiotensin II (AngII), chromatin-bound myocyte enhancer factor 2 (MEF2), acetylated histone H4 (H4ac), and H3K27ac; upregulation of hypertrophy-related genes; and decreased levels of histone deacetylase (HDAC) 4 and HDAC5 bound to the promoters of hypertrophy-related genes. Furthermore, incubation of atrial cardiomyocytes with AngII increased their cross-sectional area and improved the expression of hypertrophy-related genes. AngII also promoted the phosphorylation of HDAC4 and HDAC5 and induced their nuclear export. RNA sequencing analyses revealed that AngII significantly upregulated genes associated with cardiac hypertrophy. Chromatin immunoprecipitation showed that this correlated with increased levels of chromatin-bound MEF2, H4ac, and H3K27ac and decreased HDAC4 and HDAC5 enrichment in the promoters of hypertrophy-related genes. Moreover, these AngII-induced prohypertrophic effects could be partially reverted by treatment with the AngII receptor blocker losartan. Conclusions AngII had a prohypertrophic effect on atrial cardiomyopathy which was epigenetic-dependent. Patients with atrial fibrillation manifest an increased susceptibility to hypertrophy and exhibit epigenetic characteristics that are permissive for the transcription of hypertrophy-related genes. AngII induces histone acetylation via the cytoplasmic-nuclear shuttling of HDACs, which constitutes a novel mechanism of atrial hypertrophy regulation and might provide a promising therapeutic strategy for atrial cardiomyopathy.

Abstract 3708: OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates tumors in combination with PD-1 inhibition

Abstract Post-translational modifications of DNA and histones can improve the intrinsic antitumor capacity of the immune system. Using the iOTarg screening platform, salt-inducible kinase 3 (SIK3) was identified as a novel epigenetic modulator in cancer therapy. SIK3, a serine/threonine kinase of the AMP-activated protein kinase family, is known for regulating the NF-κB driven gene landscape through phosphorylation of class IIa histone deacetylases (HDACs) and CREB-regulated transcriptional coactivators causing the tumor to evade death receptor-mediated killing. Moreover, salt inducible kinases were recently described to regulate cell cycle checkpoints and thereby promote cancer cell proliferation. We demonstrate that SIK3 knockdown abates downstream pro-survival signaling and induces cell death in a distinct panel of tumor cell lines. OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 was shown to effectively reduce TNF-induced HDAC4 phosphorylation and downstream NF-κB activity in a dose-dependent manner, thereby enhancing apoptosis in murine and human tumor cell lines. OMX-0407 dose-dependent suppression of intratumoral NF-κB activity was shown in vivo in an MC38 NF-κB-luc reporter cell line. Using OMX-0407 monotherapy, this translated to significant tumor growth inhibition as well as prolonged survival in the highly immune infiltrated syngeneic murine colorectal carcinoma model MC38. Besides its direct inhibitory effects on cancer cells, OMX-0407 repolarizes the tumor microenvironment (TME) by strongly decreasing regulatory T cells (T-regs) and M2-polarized macrophages in the tumor bed, while not affecting the peripheral T cell compartment. Thus, exposure to OMX-0407 leads to a distinct pro-inflammatory TME, characterized by an expansion of activated cytotoxic T lymphocytes (CTL) and an increased CTL-to-T-reg ratio. Using an immune-excluded tumor phenotype as seen in the breast cancer mouse model EMT6, we demonstrated that OMX-0407 and anti-PD-1 act synergistically by combining the sensitization towards cell death with a reduction in immunosuppressive TME and an increase in cytotoxic T cell activity, despite having only minimal anti-tumor efficacy as monotherapy. Thereby, partial or complete tumor remission in 60% of the animals and extension of overall survival were achieved. In summary, OMX-0407, a first-in-class oral SIK3 inhibitor, demonstrates potent monotherapy efficacy in a pro-inflammatory tumor setting by reshaping the immune compartment and accelerating tumor cell death. The ability of OMX-0407 to remodel an immunosuppressed TME in a generally cold tumor setting, harbors great clinical potential for OMX-0407 combination therapy with anti-PD-1/PD-L1 immune checkpoint blockade, specifically in patients with high unmet medical need who are resistant to current immune checkpoint inhibitor monotherapy. Citation Format: Christina Hartl, Ilona-Petra Maser, Tillmann Michels, Ronny Milde, Vanessa Klein, Philipp Beckhove, Nisit Khandelwal, Hannes Loferer, Stefan Bissinger. OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates tumors in combination with PD-1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3708.

Engineered synthetic cell penetrating peptide with intracellular anti-inflammatory bioactivity: An in vitro and in vivo study.

Various biomaterials have been used for bone and cartilage regeneration, and inflammation associated with biomaterial implantation is also increased. A 15-mer synthetic anti-inflammatory peptide (SAP15) was designed from human β-defensin 3 to penetrate cells and induce intracellular downregulation of inflammation. The downregulation of inflammation was achieved by the binding of SAP15 to intracellular histone deacetylase (HDAC5). SAP15-mediated inhibition of inflammation was examined in vitro and in vivo using murine macrophages, human articular chondrocytes, and a collagen-induced arthritis (CIA) rat model. Surface plasmon resonance and immunoprecipitation assays indicated that SAP15 binds to HDAC5. SAP15 inhibited the lipopolysaccharide (LPS)-induced phosphorylation of intracellular HDAC5 and NF-κB p65 in murine macrophages. SAP15 treatment increased aggrecan and type II collagen expression and decreased osteocalcin expression in LPS-induced chondrocytes. Subcutaneous injection of SAP15-loaded sodium hyaluronic acid (HA) solution significantly decreased hind paw swelling, joint inflammation, and serum cytokine levels in CIA rats compared with the effects of sodium HA solution alone. The SAP15-loaded HA group exhibited preservation of cartilage and bone structure in CIA rat joints. Moreover, a more robust anti-inflammatory effect of the SAP15 loaded HA was observed than that of etanercept (an anti-tumor necrosis factor-alpha [TNF-α] antibody)-loaded HA. These findings suggest that SAP15 has an anti-inflammatory effect that is not controlled by sodium HA and is mediated by inhibiting HDAC5, unlike the anti-inflammatory mechanism of etanercept. These results demonstrate that SAP15 is useful as an inflammatory regulator of biomaterials and can be developed as a therapeutic for the treatment of inflammation.

The role of G protein-coupled receptor kinase 4 in cardiomyocyte injury after myocardial infarction.

G protein-coupled receptor kinase 4 (GRK4) has been reported to play an important role in hypertension, but little is known about its role in cardiomyocytes and myocardial infarction (MI). The goal of present study is to explore the role of GRK4 in the pathogenesis and progression of MI. We studied the expression and distribution pattern of GRK4 in mouse heart after MI. GRK4 A486V transgenic mice, inducible cardiomyocyte-specific GRK4 knockout mice, were generated and subjected to MI with their control mice. Cardiac infarction, cardiac function, cardiomyocyte apoptosis, autophagic activity, and HDAC4 phosphorylation were assessed. The mRNA and protein levels of GRK4 in the heart were increased after MI. Transgenic mice with the overexpression of human GRK4 wild type (WT) or human GRK4 A486V variant had increased cardiac infarction, exaggerated cardiac dysfunction and remodelling. In contrast, the MI-induced cardiac dysfunction and remodelling were ameliorated in cardiomyocyte-specific GRK4 knockout mice. GRK4 overexpression in cardiomyocytes aggravated apoptosis, repressed autophagy, and decreased beclin-1 expression, which were partially rescued by the autophagy agonist rapamycin. MI also induced the nuclear translocation of GRK4, which inhibited autophagy by increasing HDAC4 phosphorylation and decreasing its binding to the beclin-1 promoter. HDAC4 S632A mutation partially restored the GRK4-induced inhibition of autophagy. MI caused greater impairment of cardiac function in patients carrying the GRK4 A486V variant than in WT carriers. GRK4 increases cardiomyocyte injury during MI by inhibiting autophagy and promoting cardiomyocyte apoptosis. These effects are mediated by the phosphorylation of HDAC4 and a decrease in beclin-1 expression.

KR-39038, a Novel GRK5 Inhibitor, Attenuates Cardiac Hypertrophy and Improves Cardiac Function in Heart Failure.

G protein-coupled receptor kinase 5 (GRK5) has been considered as a potential target for the treatment of heart failure as it has been reported to be an important regulator of pathological cardiac hypertrophy. To discover novel scaffolds that selectively inhibit GRK5, we have identified a novel small molecule inhibitor of GRK5, KR-39038 [7-((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)-2-(2-chlorophenyl)-6-fluoroquinazolin-4(3H)-one]. KR-39038 exhibited potent inhibitory activity (IC50 value=0.02 µM) against GRK5 and significantly inhibited angiotensin II-induced cellular hypertrophy and HDAC5 phosphorylation in neonatal cardiomyocytes. In the pressure overload-induced cardiac hypertrophy mouse model, the daily oral administration of KR-39038 (30 mg/kg) for 14 days showed a 43% reduction in the left ventricular weight. Besides, KR-39038 treatment (10 and 30 mg/kg/day, p.o.) showed significant preservation of cardiac function and attenuation of myocardial remodeling in a rat model of chronic heart failure following coronary artery ligation. These results suggest that potent GRK5 inhibitor could effectively attenuate both cardiac hypertrophy and dysfunction in experimental heart failure, and KR-39038 may be useful as an effective GRK5 inhibitor for pharmaceutical applications.

Abstract 6698: A first-in-class SIK3 inhibitor, OMX-0370, effectively inhibits tumor growth in syngeneic tumor models, as single agent, by abolishing tumor resistance to immune-derived TNF

Abstract SIK3 is an intracellular serine/threonine kinase belonging to the AMPK superfamily. We recently discovered a novel role of SIK3 in conferring TNF resistance to tumor cells. Resistance to TNF is an emerging mode of immune evasion in multiple solid tumors. In fact, while treating a broad panel of tumor cell lines with TNF we observed that almost 70% were either resistant or even proliferative in response to TNF. SIK3 knockout using CRISPR re-sensitized human PANC-1 and murine MC38 tumor cells to TNF-mediated death. Intratumoral SIK3 induces TNF resistance by retaining HDAC4 in the cytoplasm thus keeping the chromatin open and potentiating the TNF-driven pro-tumorigenic activity of NF-κB. To translate these findings into the clinic, we have developed a potent (low nM range) inhibitor of SIK3, OMX-0370. Firstly, OMX-0370 exhibits dose-dependent inhibition of HDAC4 phosphorylation and nuclear NF-κB activity in response to TNF, thereby effectively neutralizing the SIK3-HDAC4-NF-κB axis. Consequently, treatment of human as well as murine tumor cells with OMX-0370 induces significant TNF-mediated apoptosis while sparing non-TNF treated cells. In mouse DMPK studies, OMX-0370 was found to be orally bioavailable with a favorable pharmacokinetic profile and was well tolerated at 100 mg/kg twice daily dosing in wild type C57BL/6 mice. Having shown a potent and favorable biochemical, functional as well as pharmacokinetic profile of the molecule, we next investigated the ability of OMX-0370 to inhibit the growth of established tumors in multiple syngeneic tumor models, including MC38, EMT6 and RENCA. Notably, OMX-0370 showed significant tumor growth inhibition as a single agent, which was found to be even superior to anti-PD-1 antibody treatment in RENCA and EMT-6 models. Immune profiling showed enhanced activation of intratumoral T cells, improved ratio of CTLs to Tregs and depletion of tumor-associated M2 macrophages, while no effect on peripheral leukocyte counts was observed. To monitor target engagement and pharmacodynamics of OMX-0370 in vivo, we developed a reporter MC38 cell line expressing luciferase under a NF-κB promotor. Using this reporter cell line, we could show that OMX-0370 inhibits TNF-induced NF-κB activation in a dose-dependent manner. Encouraged by the strong single-agent activity of OMX-0370 in solid tumor models, we have developed follow-on variants that exhibit higher potency as well as improved exposure in vivo. In summary, we here report that OMX-0370, a first-in-class inhibitor of SIK3 kinase, is an active immunotherapeutic drug in vivo which effectively abolishes the TNF-driven NF-κB activity in tumors and re-sensitize them to TNF-induced apoptosis. OMX-0370 and its follow-on improved variants address the high unmet medical need of effective immunotherapeutic agents that neutralize clinically relevant and key orthogonal immune evasion axes in solid tumors as a monotherapy regimen. Citation Format: Tillmann Michels, Stefan Bissinger, Peter Sennhenn, Hannes Loferer, Catarina Martins Freire, Olivia Reidell, Sebastian Meier-Ewert, Apollon Papadimitriou, Philipp Beckhove, Nisit Khandelwal. A first-in-class SIK3 inhibitor, OMX-0370, effectively inhibits tumor growth in syngeneic tumor models, as single agent, by abolishing tumor resistance to immune-derived TNF [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6698.

Oxidation of HDAC4 by Nox4-derived H2O2 maintains tube formation by endothelial cells

NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces H2O2 which can directly oxidize cysteine residues. With this post-translational modification, activity, stability, localization and protein-protein interactions of the affected protein is altered. Nox4 controls differentiation, cellular homeostasis and prevents inflammation. Therefore, is likely that epigenetic mechanisms contribute to the effects of Nox4. One group of epigenetic modifiers are class IIa histone deacetylases (HDACs). We hypothesize that Nox4-derived H2O2 oxidizes HDACs and analyzed whether HDACs can be differentially oxidized by Nox4. As an artificial system, we utilized HEK293 cells, overexpressing Nox4 in a tetracycline-inducible manner. HDAC4 was oxidized upon Nox4 overexpression. Additionally, Nox4 overexpression increased HDAC4 phosphorylation on Ser632. H2O2 disrupted HDAC4/Mef2A complex, which de-represses Mef2A. In endothelial cells such as HUVECs and HMECs, overexpression of HDAC4 significantly reduced tube formation. Overexpression of a redox insensitive HDAC4 had no effect on endothelial tube formation. Treatment with H2O2, induction of Nox4 expression by treatment of the cells with TGFβ and co-overexpression of Nox4 not only induced phosphorylation of HDAC4, but also restored the repressive effect of HDAC4 for tube formation, while overexpression of a redox dead mutant of Nox4 did not.Taken together, Nox4 oxidizes HDAC4, increases its phosphorylation, and eventually ensures proper tube formation by endothelial cells.

Cyclic AMP represses pathological MEF2 activation by myocyte-specific hypo-phosphorylation of HDAC5

Class IIa histone deacetylases (HDACs) critically regulate cardiac function through the repression of the activity of myocyte enhancer factor 2 (MEF2)-dependent gene programs. Protein kinase D (PKD) and Ca2+/Calmodulin-dependent kinase II (CaMKII) activate MEF2 by phosphorylating distinct HDAC isoforms and thereby creating 14–3-3 binding sites for nucleo-cytoplasmic shuttling. Recently, it has been shown that this process is counteracted by cyclic AMP (cAMP)-dependent signaling. Here, we investigated the specific mechanisms of how cAMP-dependent signaling regulates distinct HDAC isoforms and determined their relative contributions to the protection from pathological MEF2 activation. We found that cAMP is sufficient to induce nuclear retention and to blunt phosphorylation of the 14–3-3 binding sites of HDAC5 (Ser259/498) and HDAC9 (Ser218/448) but not HDAC4 (Ser246/467/632). These regulatory events could be observed only in cardiomyocytes and myocyte-like cells but not in non-myocytes, pointing to an indirect myocyte-specific mode of action. Consistent with one previous report, we found that blunted phosphorylation of HDAC5 and HDAC9 was mediated by protein kinase A (PKA)-dependent inhibition of PKD. However, we show by the use of neonatal cardiomyocytes derived from genetic HDAC mouse models that endogenous HDAC5 but not HDAC9 contributes specifically to the repression of endogenous MEF2 activity. HDAC4 contributed significantly to the repression of MEF2 activity but based on the mechanistic findings of this study combined with previous results we attribute this to PKA-dependent proteolysis of HDAC4. Consistently, cAMP-induced repression of agonist-driven cellular hypertrophy was blunted in cardiomyocytes deficient for both HDAC5 and HDAC4. In conclusion, cAMP inhibits MEF2 through both nuclear accumulation of hypo-phosphorylated HDAC5 and through a distinct HDAC4-dependent mechanism.

GSK3β inhibits HDAC4 to Alleviate Intravertebral Disc Degeneration Through KLF5 and ASK1 Downregulation

Intervertebral disc degeneration (IDD) is a major cause of lower back pain, yet the molecular mechanisms are not fully understood. Therefore, the effects of HDAC4 and its upstream and downstream signaling molecules on mediating IDD are determined in this study. IDD mouse model was established by injury of the vertebrate. Nucleus pulposus (NP) cells were collected for culture and transfection. Disc height index (DHI) was determined by X-ray. Pain in IDD mice was evaluated by mechanical and thermal sensitivity testes. Interaction between GSK3β and HDAC4 was assessed by co-immunoprecipitation. The effect of HDAC4 on KLF5 acetylation was determined by immunoprecipitation. Promoter region binding was assessed by chromatin immunoprecipitation. NP cells apoptosis was determined by TUNEL assay. Degradation of extracellular matrix was studied by safranin O fast green staining. Morphology of intervertebral disc tissue was determined by HE staining. mRNA and protein expression of GSK3β, HDAC4, phosphorylated HDAC4, KLF5, and ASK1 were determined by qRT-PCR and Western blotting, respectively. HDAC4 was upregulated while GSK3β was downregulated in IDD mice. HDAC4 silencing alleviated IDD. GSK3β promoted HDAC4 phosphorylation and degradation. HDAC4 promoted ASK1 expressed through, leading to enhanced IDD. GSK3β overexpression increased DHI, inhibited NP cell apoptosis, alleviated disc degeneration, and increased mechanical and thermal pain thresholds in IDD mice. The addition of HDAC4 overexpression reversed these effects of GSK3β. In conclusion, HDAC4 was degraded by GSK3β, leading to reduced IDD through KLF5 and ASK1 downregulation. Funding Statement: None. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The above experiments were approved by the Animal Ethics Committee in Guangxi University of Chinese Medicine, Guangxi International Zhuang Medicine Hospital.

The myokine meteorin-like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2.

Meteorin‐like (metrnl) is a recently identified adipomyokine that beneficially affects glucose metabolism; however, its underlying mechanism of action is not completely understood. We here show that the level of metrnl increases in vitro under electrical pulse stimulation and in vivo in exercised mice, suggesting that metrnl is secreted during muscle contractions. In addition, metrnl increases glucose uptake via the calcium‐dependent AMPKα2 pathway in skeletal muscle cells and increases the phosphorylation of HDAC5, a transcriptional repressor of GLUT4, in an AMPKα2‐dependent manner. Phosphorylated HDAC5 interacts with 14‐3‐3 proteins and sequesters them in the cytoplasm, resulting in the activation of GLUT4 transcription. An intraperitoneal injection of recombinant metrnl improved glucose tolerance in mice with high‐fat‐diet‐induced obesity or type 2 diabetes, but not in AMPK β1β2 muscle‐specific null mice. Metrnl improves glucose metabolism via AMPKα2 and is a promising therapeutic candidate for glucose‐related diseases such as type 2 diabetes.

THE ROLE OF G PROTEIN-COUPLED RECEPTOR KINASE 4 IN CARDIOMYOCYTE INJURY AFTER MYOCARDIAL INFARCTION

Objective: G-protein-coupled receptor kinase 4 (GRK4) has been reported to play a regulatory role in hypertension, but little is known of its role in cardiomyocytes and myocardial infarction (MI). The goal of present study is to explore the role of cardiac-restricted GRK4 overexpression in the pathogenesis and progression of MIDesign and method: GRK4 WT/ A486 V transgenic mice and GRK4 cardiac specific knocked out mice were generated and subjected to MI surgery. Then the survival, scar size, cardiac function and cardiomyocyte apoptosis were examined. The LC3 B protein level and GFP-LC3B were assessed to determine the autophagic activity in different groups. The phosphorylation resistant mutant HDAC4 S3A were used to investigated the how GRK4 affect autophagy. Con-focal microscope and co-immunoprecipitation were performed to reveal the interaction between GRK4 and HDAC4. We also investigated the cardiac function of MI patient between GRK4 WT and A486 V carrier. Results: The mRNA and protein expressions of GRK4 were increased in heart tissue injured by MI injury. Mice with cardiac overexpression of human GRK4 wild-type or human GRK4–486A > V were vulnerable to MI, evidenced by exaggerated cardiomyocyte apoptosis and cardiac dysfunction and remodeling. By contrast, the MI-induced cardiac dysfunction and remodeling were reduced in GRK4 knockout mice. GRK4 overexpression in cardiomyocytes had aggravated apoptosis, decreased autophagy, and decreased beclin1 expression, which were ameliorated by the autophagy agonist rapamycin. MI also induced the nuclear aggregation of GRK4. GRK4 inhibited autophagy by decreasing HDAC4 nuclear localization. A constitutively nuclear-localized mutant HDAC4 S3A partially restored the GRK4-induced inhibition of autophagy. Patients with MI carrying the constitutively active GRK4 486A > V variant had greater impairment of cardiac function than wild-type carriers. Conclusions: GRK4 increases cardiomyocyte injury caused by MI by inhibiting autophagy and promoting cardiomyocyte apoptosis. These GRK4 effects are mediated by phosphorylation of HDAC4 and a decrease in beclin1 expression.

Yin-Yang 1 transcription factor modulates ST2 expression during adverse cardiac remodeling post-myocardial infarction

BackgroundThe cardioprotective effects of metformin remain poorly defined. Interleukin (IL)-33/ST2L signaling is a novel cardioprotective pathway, which is antagonized by the soluble isoform sST2. No data exist about the regulation of ST2 expression. This study aimed to evaluate the pathophysiological implication of Yin-Yang 1 (Yy1) transcription factor in cardiac remodeling and the expression of the soluble ST2 isoform. Methods and resultsMyocardial infarction (MI) was induced in Wistar rats randomly receiving metformin or saline solution by permanent ligation of the left anterior coronary artery. In addition, a model of cardiomyocyte “biochemical strain” was used. Metformin administration improved post-MI cardiac remodeling, an effect that was associated with increased IL-33 and reduced sST2 levels in the myocardium. The anti-remodeling effects of metformin were also associated with a decrease in the transcription factor Yy1 intranuclear level and lower levels of phosphorylated HDAC4 within the cytoplasmic space. These effects were also observed in a cardiomyocyte biochemical strain model, where Yy1 silencing or HDAC4 inhibition blocked sST2 production in cardiomyocytes. Metformin blocked the HDAC4 phosphorylation induced by MI, preventing its export from the nucleus to the cytosol. The presence of dephosphorylated HDAC4 in the nucleus acted as a co-repressor of Yy1, repressing sST2 expression. ConclusionThe transcription factor Yy1 regulates sST2 expression, and repression of Yy1 by metformin results in lower levels of sST2 that are associated with favorable myocardial remodeling. The manipulation of YY1 or its co-repressor HDAC4 emerge as new targets to modulate ST2/IL33 signaling and prevent adverse cardiac remodeling.