Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation.

Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.

Muscle-Specific Pyruvate Kinase Isoforms, Pkm1 and Pkm2, Regulate Mammalian SWI/SNF Proteins and Histone 3 Phosphorylation During Myoblast Differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, Pkm1 and Pkm2, function in glycolysis, but Pkm2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of Pkm1 and Pkm2 during myoblast differentiation. RNA-seq analysis revealed that Pkm2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. Dpf2 and Baf250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for activation of myogenic gene expression during differentiation. Pkm2 also mediated the incorporation of Dpf2 and Baf250a into the regulatory sequences controlling myogenic gene expression. Pkm1 did not affect expression but was required for nuclear localization of Dpf2. Additionally, Pkm2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters, but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for Pkm2 and a novel function for Pkm1 in gene expression and chromatin regulation during myoblast differentiation.

Single nucleotide polymorphisms and Zn transport by ZIP11 shape functional phenotypes of HeLa cells.

Zinc (Zn) is a vital micronutrient with essential roles in biological processes like enzyme function, gene expression, and cell signaling. Disruptions in the cellular regulation of Zn2+ ions often lead to pathological states. Mammalian Zn transporters, such as ZIP11, play a key role in homeostasis of this ion. ZIP11 resides predominately in the nucleus and Golgi apparatus. Our laboratory reported a function of ZIP11 in maintaining nuclear Zn levels in HeLa cervical cancer cells. Analyses of cervical and ovarian cancer patients' datasets identified four coding, single nucleotide polymorphisms (SNPs) in SLC39A11, the gene that encodes ZIP11, correlating with disease severity. We hypothesized that these SNPs might translate to functional changes in the ZIP11 protein by modifying access to substrate availability. We also proposed that a metal-binding site (MBS) in ZIP11 is crucial for transmembrane Zn2+ transport and required for maintenance of various pathogenic phenotypes observed in HeLa cells. Here, we investigated these claims by re-introducing single the SLC39A11 gene encoding for mutant residues associated with the SNPs, as well as MBS mutations into HeLa cells knocked down for the transporter. Some SNPs-encoding ZIP11 variants rescued Zn levels, proliferation, migration, and invasiveness of knockdown (KD) cells. Conversely, single MBS mutations mimicked the traits of KD cells, confirming the transporter's role in establishing and maintaining proliferative, migratory, and invasive traits. Overall, the intricate role of Zn in cellular dynamics and cancer progression underscores the significance of Zn transporters like ZIP11 in potential therapeutic interventions.

PRMT5 orchestrates EGFR and AKT networks to activate NFκB and promote EMT.

Neuroblastoma remains a formidable challenge in pediatric oncology, representing 15% of cancer-related mortalities in children. Despite advancements in combinatorial and targeted treatments improving survival rates, nearly 50% of patients with high-risk neuroblastoma will ultimately succumb to their disease. Dysregulation of the epithelial-mesenchymal transition (EMT) is a key mechanism of tumor cell dissemination, resulting in metastasis and poor outcomes in many cancers. Our prior work identified PRMT5 as a key regulator of EMT via methylation of AKT at arginine 15, enhancing the expression of EMT-driving transcription factors and facilitating metastasis. Here, we identify that PRMT5 directly regulates the transcription of the epidermal growth factor receptor (EGFR). PRMT5, through independent modulation of the EGFR and AKT pathways, orchestrates the activation of NFκB, resulting in the upregulation of the pro-EMT transcription factors ZEB1, SNAIL, and TWIST1. Notably, EGFR and AKT form a compensatory feedback loop, reinforcing the expression of these EMT transcription factors. Small molecule inhibition of PRMT5 methyltransferase activity disrupts EGFR/AKT signaling, suppresses EMT transcription factor expression and ablates tumor growth in vivo . Our findings underscore the pivotal role of PRMT5 in the control of the EMT program in high-risk neuroblastoma.

Implications of the 375W mutation for HIV-1 tropism and vaccine development.

Understanding how different amino acids affect the HIV-1 envelope (Env) trimer will greatly help the design and development of vaccines that induce broadly neutralizing antibodies (bnAbs). A tryptophan residue at position 375 that opens the CD4 binding site without modifying the trimer apex was identified using our saturation mutagenesis strategy. 375W was introduced into a large panel of 27 transmitted/founder, acute stage, chronic infection, and AIDS macrophage-tropic and non-macrophage-tropic primary envelopes from different clades (A, B, C, D, and G) as well as complex and circulating recombinants. We evaluated soluble CD4 and monoclonal antibody neutralization of WT and mutant Envs together with macrophage infection. The 375W substitution increased sensitivity to soluble CD4 in all 27 Envs and macrophage infection in many Envs including an X4 variant. Importantly, 375W did not impair or abrogate neutralization by potent bnAbs. Variants that were already highly macrophage tropic were compromised for macrophage tropism, indicating that other structural factors are involved. Of note, we observed a macrophage-tropic (clade G) and intermediate macrophage-tropic (clades C and D) primary Envs from the blood and not from the central nervous system (CNS), indicating that such variants could be released from the brain or evolve outside the CNS. Our data also indicate that "intermediate" macrophage-tropic variants should belong to a new class of HIV-1 tropism. These Envs infected macrophages more efficiently than non-macrophage-tropic variants without reaching the high levels of macrophage-tropic brain variants. In summary, we show that 375W is ideal for inclusion into HIV-1 vaccines, increasing Env binding to CD4 for widely diverse Envs from different clades and disease stages.IMPORTANCESubstitutions exposing the CD4 binding site (CD4bs) on HIV-1 trimers but still occluding non-neutralizing, immunogenic epitopes are desirable to develop HIV-1 vaccines. If such substitutions induce similar structural changes in trimers across diverse clades, they could be exploited for the development of multi-clade envelope (Env) vaccines. We show that the 375W substitution increases CD4 affinity for envelopes of all clades, circulating recombinant forms, and complex Envs tested, independent of disease stage. Clade B and C Envs with an exposed CD4bs were described for macrophage-tropic strains from the central nervous system (CNS). Here, we show that intermediate (clades C and D) and macrophage-tropic (clade G) envelopes can be detected outside the CNS. Vaccines targeting the CD4bs will be particularly effective against such strains and CNS disease.

PRMT5 activates AKT via methylation to promote tumor metastasis.

Protein arginine methyltransferase 5 (PRMT5) is the primary methyltransferase generating symmetric-dimethyl-arginine marks on histone and non-histone proteins. PRMT5 dysregulation is implicated in multiple oncogenic processes. Here, we report that PRMT5-mediated methylation of protein kinase B (AKT) is required for its subsequent phosphorylation at Thr308 and Ser473. Moreover, pharmacologic or genetic inhibition of PRMT5 abolishes AKT1 arginine 15 methylation, thereby preventing AKT1 translocation to the plasma membrane and subsequent recruitment of its upstream activating kinases PDK1 and mTOR2. We show that PRMT5/AKT signaling controls the expression of the epithelial-mesenchymal-transition transcription factors ZEB1, SNAIL, and TWIST1. PRMT5 inhibition significantly attenuates primary tumor growth and broadly blocks metastasis in multiple organs in xenograft tumor models of high-risk neuroblastoma. Collectively, our results suggest that PRMT5 inhibition augments anti-AKT or other downstream targeted therapeutics in high-risk metastatic cancers.

HPV16-E6 Oncoprotein Activates TGF-ß and Wnt/ß-Catenin Pathways in the Epithelium-Mesenchymal Transition of Cataracts in a Transgenic Mouse Model.

OBJECTIVE: This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-ß and Wnt/ß-catenin signaling activation. MATERIALS AND METHODS: Cataracts of K14E6 mice were analysed histologically; and components of TGF-ß and Wnt/ß-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor. RESULTS: The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses' cell morphology were due to actin filaments reorganisation, activation of TGF-ß and Wnt/ß-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels. CONCLUSION: The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-ß and Wnt/ß-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.

The PDZ-Binding Motif of HPV16-E6 Oncoprotein Modulates the Keratinization and Stemness Transcriptional Profile In Vivo.

OBJECTIVE: The aim of this work was to compare the early gene expression profiles in the skin of HPV16-E6 transgenic mice regulated by the E6 PDZ-binding motif. MATERIALS AND METHODS: The global transcriptional profiles in dorsal skin biopsies from K14E6 and K14E6Δ146-151 transgenic mice were compared using microarrays. Relevant genes obtained from the most differentially expressed processes were further examined by RT-qPCR, in situ RT-PCR, Western blot, or immunofluorescence. RESULTS: The transcriptomic landscape of K14E6 versus K14E6Δ146-151 shows that the most affected expression profiles were those related to keratinocyte differentiation, stem cell maintenance, and keratinization. Additionally, downregulation of epidermal stemness markers such as K15 and CD34, as well as the upregulation of cytokeratin 6b, appeared to be dependent on the E6 PDZ-binding motif. Finally, wound healing, a physiological process linked to stemness, is impaired in the K14E6 mice compared to K14E6Δ146-151. CONCLUSION: The E6 PDZ-binding motif appears to affect stemness and keratinization during early stages of skin carcinogenesis. As E6 plays a significant role in HPV-induced skin carcinogenesis, the K14E6 versus K14E6Δ146-151 transcriptional profile provides a source of valuable data to uncover novel E6 functions in the skin.

Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells.

BACKGROUND/AIMS: The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. METHODS: We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. RESULTS: Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. CONCLUSION: Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.

EGF regulates claudin-2 and -4 expression through Src and STAT3 in MDCK cells.

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.