Gabriela Calounova

Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Shb (Src homology 2 protein B) is an adapter protein downstream of the vascular endothelial growth factor receptor receptor-2 (VEGFR-2). Previous experiments have suggested a role for Shb in endothelial cell function. Recently, the Shb gene was inactivated and Shb null mice were obtained on a mixed genetic background, but not on C57Bl6 mice. The present study was performed to address endothelial function in the Shb knockout mouse and its relevance for tumor angiogenesis. Tumor growth was retarded in Shb mutant mice, and this correlated with decreased angiogenesis both in tumors and in Matrigel plugs. Shb null mice display an abnormal endothelial ultrastructure in liver sinusoids and heart capillaries with cytoplasmic extensions projecting toward the lumen. Shb null heart VE-cadherin staining was less distinct than that of control heart, exhibiting in the former case a wavy and punctuate pattern. Experiments on isolated endothelial cells suggest that these changes could partly reflect cytoskeletal abnormalities. Vascular permeability was increased in Shb null mice in heart, kidney, and skin, whereas VEGF-stimulated vascular permeability was reduced in Shb null mice. It is concluded that Shb plays an important role in maintaining a functional vasculature in adult mice, and that interference with Shb signaling may provide novel means to regulate tumor angiogenesis.

Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero

SHB is an Src homology 2 domain‐containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb−/− pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb‐ gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/−) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb−/− animals, but increased number of Shb+/− animals. The Shb− allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb−/− offspring. Shb−/− animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/− female mice ovulated preferentially Shb− oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development. Developmental Dynamics 236:2485–2492, 2007.

Shb deficient mice display an augmented TH2 response in peripheral CD4+ T cells

BackgroundShb, a ubiquitously expressed Src homology 2 domain-containing adaptor protein has previously been implicated in the signaling of various tyrosine kinase receptors including the TCR. Shb associates with SLP76, LAT and Vav, all important components in the signaling cascade governing T cell function and development. A Shb knockout mouse was recently generated and the aim of the current study was to address the importance of Shb deficiency on T cell development and function.ResultsShb knockout mice did not display any major changes in thymocyte development despite an aberrant TCR signaling pattern, including increased basal activation and reduced stimulation-induced phosphorylation. The loss of Shb expression did however affect peripheral CD4+ TH cells resulting in an increased proliferative response to TCR stimulation and an elevated IL-4 production of naïve TH cells. This suggests a TH2 skewing of the Shb knockout immune system, seemingly caused by an altered TCR signaling pattern.ConclusionOur results indicate that Shb appears to play an important modulating role on TCR signaling, thus regulating the peripheral CD4+ TH2 cell response.

The Src homology 2 domain-containing adapter protein B (SHB) regulates mouse oocyte maturation.

SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb −/− mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/− and −/− mice revealed impaired early embryo development in the −/− embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development.

Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner.

Abstract Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.

Impaired glucose homeostasis in Shb−/− mice

Src homology 2 domain-containing protein B (SHB) is an adapter protein involved in the regulation of beta-cell and endothelial cell function. We have recently obtained the Shb knockout mouse, and consequently, the aim of this study was to assess the effect of Shb deletion upon beta-cell function and blood glucose homeostasis. Shb-/- mice display an elevated basal blood glucose concentration, and this increase is maintained during insulin challenge in insulin sensitivity tests. To assess glucose-induced insulin secretion, pancreata were perfused, and it was observed that Shb-/- first phase insulin secretion was blunted during glucose stimulation. Gene expression of Shb-/- islets shortly after isolation was altered, with increased pancreatic and duodenal homeobox gene-1 (Pdx1) gene expression and reduced expression of Vegf-A. Islet culture normalized Pdx1 gene expression. The microvascular density of the Shb-/- islets was reduced, and islet capillary endothelial cell morphology was changed suggesting an altered microvascular function as a contributing cause to the impaired secretory activity. Capacitance measurements of depolarization-induced exocytosis indicate a direct effect on the exocytotic machinery, in particular a dramatic reduction in readily releasable granules, as responsible for the insulin-secretory defect operating in Shb-/- islets. Shb-/- mice exhibited no alteration of islet volume or beta-cell area. In conclusion, loss of Shb impairs insulin secretion, alters islet microvascular morphology, and increases the basal blood glucose concentration. The impaired insulin secretory response is a plausible underlying cause of the metabolic impairment observed in this mutant mouse.

ADAMTS: novel proteases expressed by activated mast cells.

Abstract Here we show that mast cells (MCs) express the metalloproteases of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and that ADAMTS expression is influenced by MC activation. Co-culture of MCs with live Gram-positive bacteria caused a profound induction of ADAMTS-9 and -6, as well as down-regulated expression of ADAMTS-5. Similar patterns were also seen after MC activation with calcium ionophore and by immunoglobulin E receptor crosslinking. Moreover, ADAMTS-5, -6 and -9 were all induced by activation of terminally differentiated murine peritoneal MCs and in a human MC line. ADAMTS-9 up-regulation in response to immunoglobulin E receptor crosslinking was strongly dependent on Gö6976-sensitive protein kinase C and partly dependent on nuclear factor of activated T cells and nuclear factor kappa-light-chain-enhancer of activated B cells, respectively. The expression of ADAMTS-5, -6 and -9 was closely linked to MC maturation, as shown by their strong induction during the differentiation of bone marrow precursor cells into mature MCs. ADAMTS family members have been shown to possess aggrecanase activity. Accordingly, MCs were shown to express aggrecanase activity. Finally, ADAMTS-5 protein was detected in MCs by immunocytochemistry. Taken together, the present study reveals ADAMTS expression by MCs and that MC activation regulates the expression of these proteases, thus implicating the ADAMTS family of proteases in MC function.

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells.

Background: Serglycin is a secretory granule proteoglycan with a role in intracellular storage. Results: In response to cell death-inducing agents, wild type mast cells die by apoptosis whereas serglycin−/− cells undergo necrosis. Conclusion: Serglycin promotes apoptotic versus necrotic cell death in mast cells. Significance: The present study implicates a pathway involving the secretory granule compartment in regulation of cell death. The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin−/− mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin−/− cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin−/− mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin−/− cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.

Heterogeneity among RIP-Tag2 insulinomas allows vascular endothelial growth factor-A independent tumor expansion as revealed by studies in Shb mutant mice: Implications for tumor angiogenesis

The Shb adapter protein is a signaling intermediate that operates downstream of vascular endothelial growth factor receptor‐2 (VEGFR‐2) in endothelial cells. The Shb knockout mouse displays a dysfunctional microvasculature and impaired growth of subcutaneously implanted tumor cells. We decided to investigate tumor growth and angiogenesis in the absence of Shb in an inheritable tumor model, the RIP‐Tag2 mouse, which produces insulinomas in a manner highly dependent on de novo angiogenesis. We observed a reduced tumor incidence and burden in both RIP‐Tag2 Shb−/− and RIP‐Tag2 Shb+/− mice. This correlated with a reduced microvascular density, measured as a percentage of insulinoma area positive for CD31 staining, and altered vascular morphology. However, treatment with a VEGF‐A blocking antibody was without effect on the Shb mutant tumor volume whereas it significantly inhibited tumor volume in the wild‐type mice, suggesting that in mice with reduced Shb expression tumor angiogenesis was primarily sustained by VEGF‐A independent pathway(s). This notion was further substantiated by gene expression analysis of angiogenic markers showing reduced VEGF‐A expression in Shb‐deficient tumors. Considerable heterogeneity with respect to the gene expression profiles of other angiogenic markers and the signal‐transduction characteristics was observed between different tumors, suggesting that multiple “rescue” pathways could be operating. The numbers of invasive tumors or metastases were unchanged in the Shb mutant.

Distorted Secretory Granule Composition in Mast Cells with Multiple Protease Deficiency

Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.