Abstract
The return of blood flow to ischemic heart after myocardial infarction causes ischemia–reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ...ischemia–reperfusion injury. Here we screened for targets for the treatment of ischemia–reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein–protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction.
The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the ...eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as β1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.
Polysialic acid (polySia) is a unique linear homopolymer of α2,8-linked sialic acid that has been studied extensively as a posttranslational modification of neural cell adhesion molecule in the ...central nervous system. Only two proteins are known to be polysialylated in cells of the immune system: CD56 on human natural killer cells and murine bone marrow (BM) leukocytes, and neuropilin-2 (NRP-2) on dendritic cells (DCs). We tested the hypothesis that polySia expression is regulated during maturation and migration of leukocytes and plays a role in functional activity. Using wild-type and NCAM(-/-) mice, we show that BM neutrophils express only polysialylated CD56, whereas a subset of BM monocytes expresses polysialylated CD56 and/or another polysialylated protein(s). We demonstrate that polysialylated CD56 expression is progressively down-regulated in wild-type monocytes and monocyte-derived cells during migration from BM through peripheral blood to pulmonary and peritoneal sites of inflammation. Freshly isolated monocyte-derived peritoneal macrophages are devoid of polySia yet re-express polySia on NRP-2 and an additional protein(s) after maintenance in culture. Removal of polySia from these cells enhances phagocytosis of Klebsiella pneumoniae, suggesting that down-regulation of polySia on macrophages facilitates bacterial clearance. Using wild-type and NRP-2(-/-) mice, we demonstrate that NRP-2 and an additional protein(s) are polysialylated by ST8 SiaIV in BM-derived DCs. We conclude that polySia expression in monocyte-derived cells is dynamically regulated by ST8 SiaIV activity and by expression of carrier proteins during recruitment to sites of inflammation and influences cellular interactions with microbes, contributing to innate and adaptive immune responses.
Receptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for ...expression or activity in the ischemic heart.
RTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass, was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.
In addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.
Several novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.
The presence of bone-marrow-derived stem cells was investigated in a wound-healing model where subcutaneously implanted cellulose sponges were used to induce granulation tissue formation. When ...cellulose was coated with hydroxyapatite (HA), the sponges attracted circulating haemopoietic and mesenchymal progenitor cells more efficiently than uncoated cellulose. We hypothesized that the giant cells/macrophages of HA-coated sponges recognize HA as foreign material, phagocyte or hydrolyse it and release calcium ions, which are recognized by the calcium-sensing receptors (CaRs) expressed on many cells including haemopoietic progenitors. Our results showed, indeed, that the HA-coated sponges contained more CaR-positive cells than untreated sponges. The stem cells are, most probably, responsible for the richly vascularized granulation tissue formed in HA-coated sponges. This cell-guiding property of HA-coated cellulose might be useful in clinical situations involving impaired wound repair.
The expression of a neural crest stem cell marker, polysialic acid (polySia), and its main carrier, neural cell adhesion molecule (NCAM), have been detected in some malignant tumors with high ...metastatic activity and unfavorable prognosis, but the diagnostic and prognostic value of polySia-NCAM in neuroblastoma is unclear.
A tumor tissue microarray (TMA) of 36 paraffin-embedded neuroblastoma samples was utilized to detect polySia-NCAM expression with a polySia-binding fluorescent fusion protein, and polySia-NCAM expression was compared with clinical stage, age, MYCN amplification status, histology (INPC), and proliferation index (PI).
PolySia-NCAM-positive neuroblastoma patients had more often metastases at diagnosis, and polySia-NCAM expression associated with advanced disease (P = 0.047). Most interestingly, absence of polySia-NCAM-expressing tumor cells in TMA samples, however, was a strong unfavorable prognostic factor for overall survival in advanced disease (P = 0.0004), especially when MYCN was not amplified. PolySia-NCAM-expressing bone marrow metastases were easily detected in smears, aspirates and biopsies.
PolySia-NCAM appears to be a new clinically significant molecular marker in neuroblastoma, hopefully with additional value in neuroblastoma risk stratification.
The general opinion that hemoglobin is only a carrier protein for oxygen and carbon dioxide has been challenged by several recent studies showing hemoglobin expression in other cells than those of ...the erythroid series, for example, in macrophages. We discovered β-globin expression in rat experimental granulation tissue induced by subcutaneously implanted cellulose sponges. Closer investigation revealed also α-globin expression. The first peak of the biphasic globin expression noticed during granulation tissue formation correlated with the invasion of monocytes/macrophages, whereas the second one seemed to be connected to the appearance of hematopoietic progenitors. Data presented in this study indicate globin expression both in macrophages and in immature erythroid cells as validated by erythroid-specific markers.
The growth factor Neuregulin‐1 (NRG‐1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo ...models that STAT5b mediates NRG‐1/EBBB4‐stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG‐1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG‐1‐induced cardiomyocyte hypertrophy. Dynamin‐2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin‐2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG‐1‐induced hyperplastic myocardial growth, and chemical inhibition of the Nrg‐1/Erbb4 pathway or Dynamin‐2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9‐mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG‐1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG‐1/ERBB4/STAT5b pathway in myocardial growth.
Synopsis
STAT5b regulates hypertrophic and hyperplastic growth signaling via the NRG‐1/ERBB4 pathway in cardiomyocytes at adult and embryonic stages. Patients with pathological cardiac hypertrophy show perturbed STAT5b signaling.
NRG‐1/ERBB4‐activated STAT5b induces hypertrophic growth in neonatal and adult cardiomyocytes.
Loss of stat5b in embryonic zebrafish leads to reduced hyperplastic myocardial growth and cardiac function.
Dynamin‐2 controls the cell surface localization of ERBB4, and its inhibition reduces cardiomyocyte hypertrophy.
NRG‐1/ERBB4/STAT5b signaling is perturbed in the myocardium of hypertrophic cardiomyopathy patients.
STAT5b regulates hypertrophic and hyperplastic growth signaling via the NRG‐1/ERBB4 pathway in cardiomyocytes at adult and embryonic stages. Patients with pathological cardiac hypertrophy show perturbed STAT5b signaling.
Polysialic acid, an abundant cell surface component of the developing nervous system, which declines rapidly postnatally to virtual absence in the majority of adult tissues, is highly expressed in ...some malignant tumors including neuroblastoma. We found that the binding of a noncatalytic endosialidase to polysialic acid causes internalization of the complex from the surface of neuroblastoma kSK-N-SH cells, a subline of SK-N-SH, and leads to a complete relocalization of polysialic acid to the intracellular compartment. The binding and uptake of the endosialidase is polysialic acid-dependent as it is inhibited by free excess ligand or removal of polysialic acid by active endosialidase, and does not happen if catalytic endosialidase is used in place of inactive endosialidase. A fusion protein composed of the noncatalytic endosialidase and the cytotoxic portion of diphtheria toxin was prepared to investigate whether the cellular uptake observed could be used for the specific elimination of polysialic acid-containing cells. The conjugate toxin was found to be toxic to polysialic acid-positive kSK-N-SH with an IC
of 1.0 nmol/L. Replacing the noncatalytic endosialidase with active endosialidase decreased the activity to the level of nonconjugated toxin. Normal nonmalignant cells were selectively resistant to the toxin conjugate. The results demonstrate that noncatalytic endosialidase induces a quantitative removal and cellular uptake of polysialic acid from the cell surface which, by conjugation with diphtheria toxin fragment, can be exploited for the selective elimination of polysialic acid-containing tumor cells.
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