S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

Neuroimmune/Hematopoietic Axis with Distinct Regulation by the High-Mobility Group Box 1 in Association with Tachykinin Peptides.

Hematopoiesis is tightly regulated by the bone marrow (BM) niche. The niche is robust, allowing for the return of hematopoietic homeostasis after insults such as infection. Hematopoiesis is partly regulated by soluble factors, such as neuropeptides, substance P (SP), and neurokinin A (NK-A), which mediate hematopoietic stimulation and inhibition, respectively. SP and NK-A are derived from the Tac1 gene that is alternately spliced into four variants. The hematopoietic effects of SP and NK-A are mostly mediated via BM stroma. Array analyses with 2400 genes indicated distinct changes in SP-stimulated BM stroma. Computational analyses indicated networks of genes with hematopoietic regulation. Included among these networks is the high-mobility group box 1 gene (HMGB1), a nonhistone chromatin-associated protein. Validation studies indicated that NK-A could reverse SP-mediated HMGB1 decrease. Long-term culture-initiating cell assay, with or without NK-A receptor antagonist (NK2), showed a suppressive effect of HMGB1 on hematopoietic progenitors and increase in long-term culture-initiating cell assay cells (primitive hematopoietic cells). These effects occurred partly through NK-A. NSG mice with human hematopoietic system injected with the HMGB1 antagonist glycyrrhizin verified the in vitro effects of HMGB1. Although the effects on myeloid lineage were suppressed, the results suggested a more complex effect on the lymphoid lineage. Clonogenic assay for CFU- granulocyte-monocyte suggested that HMGB1 may be required to prevent hematopoietic stem cell exhaustion to ensure immune homeostasis. In summary, this study showed how HMGB1 is linked to SP and NK-A to protect the most primitive hematopoietic cell and also to maintain immune/hematopoietic homeostasis.

A compendium of conserved cleavage and polyadenylation events in mammalian genes.

Cleavage and polyadenylation is essential for 3' end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3' UTRs and/or coding sequences. Here, we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on approximately 1.2 billion 3' end sequencing reads from more than 360 human, mouse, and rat samples. We show that ∼80% of mammalian mRNA genes contain at least one conserved PAS, and ∼50% have conserved APA events. PAS conservation generally reduces promiscuous 3' end processing, stabilizing gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with conserved APA events. Whereas tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. In addition, we show enrichment of mRNA destabilizing motifs in alternative 3' UTR sequences, leading to substantial differences in mRNA stability between 3' UTR isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Furthermore, we show that mutations of U-rich motifs around the PAS often accompany APA profile differences between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation through evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic perspective on APA functions.

Mitochondrial LonP1 protects cardiomyocytes from ischemia/reperfusion injury in vivo.

RATIONALE: LonP1 is an essential mitochondrial protease, which is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. However, the importance of LonP1 during cardiac stress is largely unknown. OBJECTIVE: To determine the functions of LonP1 during ischemia/reperfusion (I/R) injury in vivo, and hypoxia-reoxygenation (H/R) stress in vitro. METHODS AND RESULTS: LonP1 was induced 2-fold in wild-type mice during cardiac ischemic preconditioning (IPC), which protected the heart against ischemia-reperfusion (I/R) injury. In contrast, haploinsufficiency of LonP1 (LONP1+/-) abrogated IPC-mediated cardioprotection. Furthermore, LONP1+/- mice showed significantly increased infarct size after I/R injury, whereas mice with 3-4 fold cardiac-specific overexpression of LonP1 (LonTg) had substantially smaller infarct size and reduced apoptosis compared to wild-type controls. To investigate the mechanisms underlying cardioprotection, LonTg mice were subjected to ischemia (45 min) followed by short intervals of reperfusion (10, 30, 120 min). During early reperfusion, the left ventricles of LonTg mice showed substantially reduced oxidative protein damage, maintained mitochondrial redox homeostasis, and showed a marked downregulation of both Complex I protein level and activity in contrast to NTg mice. Conversely, when LonP1 was knocked down in isolated neonatal rat ventricular myocytes (NRVMs), an up-regulation of Complex I subunits and electron transport chain (ETC) activities was observed, which was associated with increased superoxide production and reduced respiratory efficiency. The knockdown of LonP1 in NRVMs caused a striking dysmorphology of the mitochondrial inner membrane, mitochondrial hyperpolarization and increased hypoxia-reoxygenation (H/R)-activated apoptosis. Whereas, LonP1 overexpression blocked H/R-induced cell death. CONCLUSIONS: LonP1 is an endogenous mediator of cardioprotection. Our findings show that upregulation of LonP1 mitigates cardiac injury by preventing oxidative damage of proteins and lipids, preserving mitochondrial redox balance and reprogramming bioenergetics by reducing Complex I content and activity. Mechanisms that promote the upregulation of LonP1 could be beneficial in protecting the myocardium from cardiac stress and limiting I/R injury.

Small Fractions of Muscular Dystrophy Embryonic Stem Cells Yield Severe Cardiac and Skeletal Muscle Defects in Adult Mouse Chimeras.

Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

Alternative cleavage and polyadenylation in spermatogenesis connects chromatin regulation with post-transcriptional control.

BACKGROUND: Most mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3' untranslated regions (3'UTRs) in testes. RESULTS: By deep sequencing of the 3' end region of poly(A) + transcripts, we report widespread shortening of 3'UTR through APA during the first wave of spermatogenesis in mouse, with 3'UTR size being the shortest in spermatids. Using genes without APA as a control, we show that shortening of 3'UTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear highly potent during spermatogenesis. We additionally found widespread regulation of APA events in introns and exons that can affect the coding sequence of transcripts and global activation of antisense transcripts upstream of the transcription start site, suggesting modulation of splicing and initiation of transcription during spermatogenesis. Importantly, genes that display significant 3'UTR shortening tend to have functions critical for further sperm maturation, and testis-specific genes display greater 3'UTR shortening than ubiquitously expressed ones, indicating functional relevance of APA to spermatogenesis. Interestingly, genes with shortened 3'UTRs tend to have higher RNA polymerase II and H3K4me3 levels in spermatids as compared to spermatocytes, features previously known to be associated with open chromatin state. CONCLUSIONS: Our data suggest that open chromatin may create a favorable cis environment for 3' end processing, leading to global shortening of 3'UTR during spermatogenesis. mRNAs with shortened 3'UTRs are relatively stable thanks to evasion of powerful mRNA degradation mechanisms acting on 3'UTR elements. Stable mRNAs generated in spermatids may be important for protein production at later stages of sperm maturation, when transcription is globally halted.

Analysis of alternative cleavage and polyadenylation by 3' region extraction and deep sequencing.

Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3' region extraction and deep sequencing (3'READS) to address mispriming issues that commonly plague poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3' ends revealed over 5,000 previously overlooked pAs (∼8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3'READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3' end-processing activity in development and differentiation.

Global ablation of the mouse Rab11a gene impairs early embryogenesis and matrix metalloproteinase secretion.

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a(null) blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a(null) embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.

Activation of PKN mediates survival of cardiac myocytes in the heart during ischemia/reperfusion.

RATIONALE: The function of PKN, a stress-activated protein kinase, in the heart is poorly understood. OBJECTIVE: We investigated the functional role of PKN during myocardial ischemia/reperfusion (I/R). METHODS AND RESULTS: PKN is phosphorylated at Thr774 in hearts subjected to ischemia and reperfusion. Myocardial infarction/area at risk (MI/AAR) produced by 45 minutes of ischemia and 24 hours of reperfusion was significantly smaller in transgenic mice with cardiac-specific overexpression of constitutively active (CA) PKN (Tg-CAPKN) than in nontransgenic (NTg) mice (15+/-5 versus 38+/-5%, P<0.01). The number of TUNEL-positive nuclei was significantly lower in Tg-CAPKN (0.3+/-0.2 versus 1.0+/-0.2%, P<0.05). Both MI/AAR (63+/-9 versus 45+/-8%, P<0.05) and the number of TUNEL-positive cells (7.9+/-1.0 versus 1.3+/-0.9%, P<0.05) were greater in transgenic mice with cardiac-specific overexpression of dominant negative PKN (Tg-DNPKN) than in NTg mice. Thr774 phosphorylation of PKN was also observed in response to H(2)O(2) in cultured cardiac myocytes. Stimulation of PKN prevented, whereas inhibition of PKN aggravated, cell death induced by H(2)O(2), suggesting that the cell-protective effect of PKN is cell-autonomous in cardiac myocytes. PKN induced phosphorylation of alpha B crystallin and increased cardiac proteasome activity. The infarct reducing effect in Tg-CAPKN mice was partially inhibited by epoxomicin, a proteasome inhibitor. CONCLUSIONS: PKN is activated by I/R and inhibits apoptosis of cardiac myocytes, thereby protecting the heart from I/R injury. PKN mediates phosphorylation of alpha B crystallin and stimulation of proteasome activity, which, in part, mediates the protective effect of PKN in the heart.

PERM1 regulates genes involved in fatty acid metabolism in the heart by interacting with PPARα and PGC-1α.

PERM1 (PGC-1/ERR-induced regulator in muscle 1) is a muscle-specific protein induced by PGC-1 and ERRs. Previous studies have shown that PERM1 promotes mitochondrial biogenesis and metabolism in cardiomyocytes in vitro. However, the role of endogenous PERM1 in the heart remains to be investigated with loss-of-function studies in vivo. We report the generation and characterization of systemic Perm1 knockout (KO) mice. The baseline cardiac phenotype of the homozygous Perm1 KO mice appeared normal. However, RNA-sequencing and unbiased pathway analyses showed that homozygous downregulation of PERM1 leads to downregulation of genes involved in fatty acid and carbohydrate metabolism in the heart. Transcription factor binding site analyses suggested that PPARα and PGC-1α are involved in changes in the gene expression profile. Chromatin immunoprecipitation assays showed that PERM1 interacts with the proximal regions of PPAR response elements (PPREs) in endogenous promoters of genes involved in fatty acid oxidation. Co-immunoprecipitation and reporter gene assays showed that PERM1 promoted transcription via the PPRE, partly in a PPARα and PGC-1α dependent manner. These results suggest that endogenous PERM1 is involved in the transcription of genes involved in fatty acid oxidation through physical interaction with PPARα and PGC-1α in the heart in vivo.

Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection.

BACKGROUND: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: (1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; (2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and (3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. RESULTS: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show (1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; (2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; (3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. CONCLUSIONS: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Development of a Novel Human CD147 Knock-in NSG Mouse Model to Test SARS-CoV-2 Viral Infection.

Background: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. Results: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD- scid IL2Rgamma null (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show 1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; 2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; 3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. Conclusions: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Development of a Novel Human CD147 Transgenic NSG Mouse Model to test SARS-CoV-2 Infection and Immune Responses.

An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the newly emerged, rapidly spreading SARS-CoV-2 and development of therapeutic strategies. Studies show that the spike (S) proteins of SARS-CoV (SARS-CoV-S-1-S) and SARS-CoV-2 (SARS-CoV-2-S) bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which is clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients); 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not develop strong clinical disease following SARS-CoV-2 infection in contrast to SARS-CoV-1. Moreover, one of most outstanding features of coronaviruses is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a receptor for SARS-CoV-2-S. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. Here we successfully generated a hCD147Tg mouse model in the NOD- scid IL2Rgamma null (NSG) background. In this hCD147Tg-NSG mouse model, the hCD147 genetic sequence was placed following the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of CD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 model. In addition, the hCD147Tg-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations with immunosuppressed conditions. The hCD147Tg-NSG mouse mode can serve as an additional animal model for interrogate whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Specific N-cadherin-dependent pathways drive human breast cancer dormancy in bone marrow.

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Recycling Endosomes in Mature Epithelia Restrain Tumorigenic Signaling.

The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, whereas suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. SIGNIFICANCE: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism.

Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy.

Activation of mammalian sterile 20-like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity-dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy.

Constitutive Expression of Inducible Cyclic Adenosine Monophosphate Early Repressor (ICER) in Cycling Quiescent Hematopoietic Cells: Implications for Aging Hematopoietic Stem Cells.

Despite extensive insights on the interaction between hematopoietic stem cells (HSCs) and the supporting bone marrow (BM) stroma in hematopoietic homeostasis there remains unanswered questions on HSC regulation. We report on the mechanism by which HSCs attain cycling quiescence by addressing a role for inducible cyclic AMP early repressor (ICER). ICER negatively transcriptional regulators of cAMP activators such as CREM and CREB. These activators can be induced by hematopoietic stimulators such as cytokines. We isolated subsets of hematopoietic cells from ten healthy donors: CD34+CD38-/c-kit + (primitive progenitor), CD34+CD38+/c-kitlow (mature progenitor) and CD34-CD38+/-/c-kitlow/- (differentiated lineage-). The relative maturity of the progenitors were verified in long-term culture initiating assay. Immunoprecipitation indicated the highest level of ICER in the nuclear extracts of CD34+/CD38- cells. Phospho (p)-CREM was also present suggesting a balance between ICER and p-CREM in HSC. ICER seems to be responsible for decrease in G1 transition, based on reduced Cdk4 protein, decreased proliferation and functional studies with propidium iodide. There were no marked changes in the cycling inhibitors, p15 and p-Rb, suggesting that ICER may act independently of other cycling inhibitors. The major effects of ICER were validated with BM mononuclear cells (BMNCs) in which ICER was ectopically expressed, and with BMNCs resistant to 5-fluorouracil- or cyclophosphamide. In total, this study ascribes a novel role for ICER in G1 checkpoint regulation in HSCs. These findings are relevant to gene therapy that require engineering of HSCs, age-related disorders that are associated with hematopoietic dysfunction and other hematological disorders.

Transgenic Expression of the Vitamin D Receptor Restricted to the Ileum, Cecum, and Colon of Vitamin D Receptor Knockout Mice Rescues Vitamin D Receptor-Dependent Rickets.

Although the intestine plays the major role in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action on calcium homeostasis, the mechanisms involved remain incompletely understood. The established model of 1,25(OH)2D3-regulated intestinal calcium absorption postulates a critical role for the duodenum. However, the distal intestine is where 70% to 80% of ingested calcium is absorbed. To test directly the role of 1,25(OH)2D3 and the vitamin D receptor (VDR) in the distal intestine, three independent knockout (KO)/transgenic (TG) lines expressing VDR exclusively in the ileum, cecum, and colon were generated by breeding VDR KO mice with TG mice expressing human VDR (hVDR) under the control of the 9.5-kb caudal type homeobox 2 promoter. Mice from one TG line (KO/TG3) showed low VDR expression in the distal intestine (<50% of the levels observed in KO/TG1, KO/TG2, and wild-type mice). In the KO/TG mice, hVDR was not expressed in the duodenum, jejunum, kidney, or other tissues. Growth arrest, elevated parathyroid hormone level, and hypocalcemia of the VDR KO mice were prevented in mice from KO/TG lines 1 and 2. Microcomputed tomography analysis revealed that the expression of hVDR in the distal intestine of KO/TG1 and KO/TG2 mice rescued the bone defects associated with systemic VDR deficiency, including growth plate abnormalities and altered trabecular and cortical parameters. KO/TG3 mice showed rickets, but less severely than VDR KO mice. These findings show that expression of VDR exclusively in the distal intestine can prevent abnormalities in calcium homeostasis and bone mineralization associated with systemic VDR deficiency.

Inducible cAMP early repressor (ICER) is a negative-feedback regulator of cardiac hypertrophy and an important mediator of cardiac myocyte apoptosis in response to beta-adrenergic receptor stimulation.

Although stimulation of the beta-adrenergic receptor increases levels of cAMP and activation of the cAMP response element (CRE) in cardiac myocytes, the role of the signaling mechanism regulated by cAMP in hypertrophy and apoptosis is not well understood. In this study we show that protein expression of inducible cAMP early repressor (ICER), an endogenous inhibitor of CRE-mediated transcription, is induced by stimulation of isoproterenol (ISO), a beta-adrenergic agonist with a peak at approximately 12 hours and persisting for more than 24 hours in neonatal rat cardiac myocytes. ICER is also upregulated by phenylephrine but not by endothelin-1. Continuous infusion of ISO also increased ICER in the rat heart in vivo. Overexpression of ICER significantly attenuated ISO- and phenylephrine-induced cardiac hypertrophy but did not inhibit endothelin-1-induced cardiac hypertrophy. Overexpression of ICER also stimulated cardiac myocyte apoptosis. Antisense inhibition of ICER significantly enhanced beta-adrenergic hypertrophy, whereas it significantly inhibited beta-adrenergic cardiac myocyte apoptosis, suggesting that endogenous ICER works as an important regulator of cardiac hypertrophy and apoptosis. Inhibition of CRE-mediated transcription by dominant-negative CRE binding protein inhibited cardiac hypertrophy, whereas it stimulated cardiac myocyte apoptosis, thereby mimicking the effect of ICER. Both ISO and ICER reduced expression of Bcl-2, an antiapoptotic molecule, whereas antisense ICER prevented ISO-induced downregulation of Bcl-2. These results suggest that ICER is upregulated by cardiac hypertrophic stimuli increasing CRE-mediated transcription in cardiac myocytes and acts as a negative regulator of hypertrophy and a positive mediator of apoptosis, in part through both inhibition of CRE-mediated transcription and downregulation of Bcl-2.

Evidence for a regulatory role of inducible cAMP early repressor in protein kinase a-mediated enhancement of vitamin D receptor expression and modulation of hormone action.

Parathyroid hormone (PTH) or activators of protein kinase A (PKA) up-regulate the vitamin D receptor (VDR) and augment the induction by 1,25-dihydroxyvitamin D(3) of the expression of target genes (24-hydroxylase and osteopontin) in osteoblastic cells. To understand regulatory mechanisms involved, we asked whether the inducible cAMP early repressor (ICER), which serves as a dominant negative regulator of cAMP-induced transcription in other endocrine systems, may similarly play a role in modulation of vitamin D hormone action. In this study we demonstrate that PTH or 8-bromo-cAMP rapidly induces ICER mRNA and protein in osteoblastic cells. In UMR 106 osteoblastic cells transfected with an expression vector containing the ICER II-gamma coding sequence, cAMP or PTH enhancement of 1,25-dihydroxyvitamin D(3)-induced osteopontin and 24-hydroxylase mRNA and transcription is inhibited. The vitamin D response element is sufficient for the PKA enhancement of VDR-mediated transcription and is also sufficient to observe the inhibitory effect of ICER. Our data indicate that the mechanism of the inhibitory effect of ICER involves an inhibition of PKA-induced VDR transcription, and this inhibition may be mediated in part by binding of ICER to a cAMP response element-like sequence in the VDR promoter. This study provides evidence for the first time that ICER has a key regulatory role in the PKA enhancement of VDR transcription and therefore in the cross-talk between the PKA signaling pathway and the vitamin D endocrine system.