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Supplementary Table 1 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Figure 3 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Figure Legends 1-7 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Table 2 Legend from 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. [Cancer Res 2009;69(5):2141–8]

Supplementary Table 2 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Figure 2 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Figure 1 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Staphylococcus aureus is a major pathogen that can cause a broad spectrum of serious infections including skin infections, pneumonia and sepsis. Peritoneal mast cells have been implicated in the host response towards various bacterial insults and to provide mechanistic insight into the role of mast cells in intraperitoneal bacterial infection we here studied the global effects of S. aureus on mast cell gene expression. After co-culture of peritoneal mast cells with live S. aureus we found by gene array analysis that they up-regulate a number of genes. Many of these corresponded to pro-inflammatory cytokines, including interleukin-3, interleukin-13 and tumour necrosis factor-α. The cytokine induction in response to S. aureus was confirmed by ELISA. To study the role of peritoneal mast cells during in vivo infection with S. aureus we used newly developed Mcpt5-Cre(+) × R-DTA mice in which mast cell deficiency is independent of c-Kit. This is in contrast to previous studies in which an impact of mast cells on bacterial infection has been proposed based on the use of mice whose mast cell deficiency is a consequence of defective c-Kit signalling. Staphylococcus aureus was injected intraperitoneally into mast-cell-deficient Mcpt5-Cre(+) × R-DTA mice using littermate mast-cell-sufficient mice as controls. We did not observe any difference between mast-cell-deficient and control mice with regard to weight loss, bacterial clearance, inflammation or cytokine production. We conclude that, despite peritoneal mast cells being activated by S. aureus in vitro, they do not influence the in vivo manifestations of intraperitoneal S. aureus infection.

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.

Granzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy.

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.

Mast cells are key effector cells in allergic reactions. When activated to degranulate, they release a plethora of bioactive compounds from their secretory granules, including mast cell-restricted proteases such as tryptase. In a previous study, we showed that tryptase, in addition to its intragranular location, can be found within the nuclei of mast cells where it truncates core histones at their N-terminal ends.Considering that the N-terminal portions of the core histones constitute sites for posttranslational modifications of major epigenetic impact, we evaluated whether histone truncation by tryptase could have an impact on epigenetic events in mast cells.Mast cells were cultured from wild-type and tryptase null mice, followed by an assessment of their profile of epigenetic histone modifications and their phenotypic characteristics.We show that tryptase truncates nucleosomal histone 3 and histone 2B (H2B) and that its absence results in accumulation of the epigenetic mark, lysine 5-acetylated H2B. Intriguingly, the accumulation of lysine 5-acetylated H2B was cell age-dependent and was associated with a profound upregulation of markers of non-mast cell lineages, loss of proliferative control, chromatin remodeling as well as extensive morphological alterations.These findings introduce tryptase-catalyzed histone clipping as a novel epigenetic regulatory mechanism, which in the mast cell context may be crucial for maintaining cellular identity.

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.

Mast cells (MCs) can have either detrimental or beneficial effects on malignant processes but the underlying mechanisms are poorly understood. Here we addressed this issue by examining the interaction between Lewis Lung Carcinoma (LLC) cells and MCs. In vivo, LLC tumors caused a profound accumulation of MCs, suggesting that LLC tumors have the capacity to attract MCs. Indeed, transwell migration assays showed that LLC-conditioned medium had chemotactic activity towards MCs, which was blocked by an antibody towards stem cell factor. In order to gain insight into the molecular mechanisms operative in tumor–MC interactions, the effect of LLC on the MC gene expression pattern was examined. As judged by gene array analysis, conditioned medium from LLC cells caused significant upregulation of numerous cell surface receptors and a pro-angiogenic Runx2/VEGF/Dusp5 axis in MCs, the latter in line with a role for MCs in promoting tumor angiogenesis. Among the genes showing the highest extent of upregulation was Tnfrsf9, encoding the anti-tumorigenic protein 4-1BB, suggesting that also anti-tumorigenic factors are induced. Quantitative RT-PCR analysis showed that 4-1BB was upregulated in a transient manner, and it was also shown that tumor cells induce 4-1BB in human MCs. Immunohistochemical analysis showed that LLC-conditioned medium induced 4-1BB also at the protein level. Together, this study provides novel insight into the molecular events associated with MC–tumor interactions and suggests that tumor cells induce both pro- and anti-tumorigenic responses in MCs.

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.

Mast cells (MC) have pathogenic roles in numerous disorders, and strategies that stabilize MC or induce MC apoptosis are therefore emerging as possible therapeutic regimens. A typical feature of MC is their high content of secretory lysosomes (granules), containing numerous components such as biogenic amines, cytokines, serglycin proteoglycan and proteases. Damage to the secretory lysosomes will thus lead to leakage of these compounds, including the proteases, into the cytosol, and this could potentially trigger apoptosis. Here, we evaluated whether MC are sensitive to cell death induced by secretory lysosome destabilization, induced by the lysosomotropic agent Leu-Leu-OMe (LLME). Human MC were sensitive to LLME-induced cell death. In contrast, fibroblasts and HEK-293 cells were largely resistant. As judged by Annexin V/propidium iodide staining, LLME caused apoptotic cell death, and this was supported by induction of caspase-3-like activity, detection of activated caspase-3 by immunoblot analysis and reduced cell death in the presence of a caspase inhibitor. In support of a role for serglycin in regulating LLME-induced cell death, the survival rate of various cell types correlated negatively with the level of serglycin expression. In summary, this study introduces the concept of using lysosomotropic agents to induce cell death of human MC.

Prader-Willi syndrome (PWS) is caused by the disturbed expression of genes from the imprinted region of 15q11-q13, but the specific contributions of individual genes remain unknown. Most paternal PWS deletions are bracketed by recurrent breakpoints BP1 or BP2 and BP3. Atypical deletions are very rare. In the present work, we describe the molecular analysis of two patients with atypical deletions using microsatellite analysis, methylation-specific MLPA, and microarray CGH. A deletion of about 2 Mb in Patient 1 started at BP2 and ended in the middle of the typically deleted region within the UBE3A gene. The deletion in Patient 2 started 1.3 Mb distal from BP2 within the C15ORF2 gene, extended over 9.5 Mb, and ended within the AVEN gene in proximal 15q14. In Patient 1 both deletion breakpoints involved repetitive regions, which precluded cloning of the junction and pointed to non-allelic homologous recombination as a possible mechanism of this rearrangement. The breakpoints in Patient 2 were sequenced, and their structure suggested non-homologous end joining as the most likely cause of this deletion. The phenotype of both patients did not depart significantly from the typical clinical picture of PWS, although some symptoms in Patient 2 were also reminiscent of the phenotype of individuals with the recently described 15q13.3 microdeletion syndrome. Our findings support previous observations of relatively mild phenotypic effects resulting from deletions that extend distally from the PWS region and observations of the modest effects of different types of genetic defects on the spectrum and severity of symptoms in PWS.

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.

Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell-mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu-Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.

Mast cells are known to have a detrimental impact on a variety of pathological conditions. There is therefore an urgent need of developing strategies that limit their harmful effects. The aim of this study was to accomplish this by developing a means of inducing mast cell apoptosis. The strategy was to identify novel compounds that induce mast cell apoptosis by permeabilization of their secretory lysosomes (granules). As a candidate, we assessed mefloquine, an anti-malarial drug that has been proposed to have lysosome-permeabilizing activity. Mefloquine was added to mast cells and administered in vivo, followed by assessment of the extent and mechanisms of mast cell death. Mefloquine was cytotoxic to murine and human mast cells. Mefloquine induced apoptotic cell death of wild-type mast cells whereas cells lacking the granule compounds serglycin proteoglycan or tryptase were shown to undergo necrotic cell death, the latter finding indicating a role of the mast cell granules in mefloquine-induced cell death. In support of this, mefloquine was shown to cause compromised granule integrity and to induce leakage of granule components into the cytosol. Mefloquine-induced cell death was refractory to caspase inhibitors but was completely abrogated by reactive oxygen species inhibition. These findings identify mefloquine as a novel anti-mast cell agent, which induces mast cell death through a granule-mediated pathway. Mefloquine may thus become useful in therapy aiming at limiting harmful effects of mast cells.

Background: Many of the functions attributed to mast cells depend on the various pro-inflammatory mediators that are secreted upon mast cell activation. These include a panel of mast cell-specific proteases. In addition, recent studies have indicated that murine mast cells also express granzyme D, a protease previously thought to be confined to cytotoxic lymphocytes. Here, we address the human relevance of the latter findings by investigating whether human mast cells express granzyme H, the granzyme that may represent the functional counterpart to murine granzyme D. Methods: Cord blood-derived mast cells, LAD2 cells and skin mast cells in situ were evaluated for their expression of granzymes using quantitative PCR, Western blot analysis and immunostaining. Mast cells were activated by either calcium ionophore stimulation or IgE receptor cross-linking. Results: Cord blood-derived mast cells and LAD2 cells were shown to express granzyme H and B mRNA, while granzyme A, K and M expression was undetectable. Mast cell activation by either calcium ionophore or IgE receptor cross-linking caused down-regulated expression of granzyme H. In contrast, granzyme B expression was up-regulated by the same stimuli. Granzyme H expression was also confirmed at the protein level, as shown by both Western blot analysis and confocal microscopy. Further, we show that granzyme H is expressed by human skin mast cells in situ. Conclusions: The present findings implicate granzyme H as a novel protease expressed by human mast cells and support earlier findings obtained in natural killer cells suggesting that granzymes B and H are reciprocally regulated.

Mast cells (MCs) are well known for their detrimental effects in the context of allergic disorders. Strategies that limit MC function can therefore have a therapeutic value. Previous studies have shown that siramesine, a sigma-2 receptor agonist originally developed as an anti-depressant, can induce cell death in transformed cells through a mechanism involving lysosomal destabilization. Since MCs are remarkably rich in lysosome-like secretory granules we reasoned that MCs might be sensitive to siramesine. Here we show that murine and human MCs are highly sensitive to siramesine. Cell death was accompanied by secretory granule permeabilization, as shown by reduced acridine orange staining and leakage of granule proteases into the cytosol. Wild type siramesine-treated MCs underwent cell death with typical signs of apoptosis but MCs lacking serglycin, a proteoglycan crucial for promoting the storage of proteases within MC secretory granules, died predominantly by necrosis. A dissection of the underlying mechanism suggested that the necrotic phenotype of serglycin(-/-) cells was linked to defective Poly(ADP-ribose) polymerase-1 degradation. In vivo, siramesine treatment of mice caused a depletion of the MC populations of the peritoneum and skin. The present study shows for the first time that MCs are highly sensitive to apoptosis induced by siramesine and introduces the possibility of using siramesine as a therapeutic agent for treatment of MC-dependent disease.

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. It is concluded that the Shb mutant background reduces tumor frequency by chronically suppressing VEGF-A dependent angiogenesis. However, VEGF-A independent angiogenesis supports a significant degree of tumor expansion in Shb-deficient mice, indicating heterogeneity in the mechanisms by which tumor expansion is promoted. Interference with Shb signaling may provide novel means for future cancer therapy.

The biological function of the Nr4a subfamily of nuclear receptors is only partially understood. Here we show for the fist time that mast cell (MC) activation processes involve the regulation of Nr4a factors. Exposure of murine bone marrow-derived MCs (BMMCs) to live bacteria causes a robust and selective upregulation of all Nr4a members (Nr4a1–Nr4a3). In response to purified LPS, strong upregulation of Nr4a3, but not of Nr4a1 or Nr4a2 was seen. Nr4a3 expression was also induced after the activation of BMMCs by IgE receptor cross-linking. Moreover, Nr4a expression was induced in activated human MCs. As shown by Western blot analysis, Nr4a phosphorylation was induced by IgE receptor cross-linking and calcium ionophore stimulation of BMMCs and LAD2 cells, respectively. By using various inhibitors of signaling pathways, Nr4a3 induction in BMMCs was shown to be strongly dependent on Go6976-sensitive kinases and partially dependent on the nuclear factor of activated T-cells (NFAT) pathway, while nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inhibition failed to inhibit Nr4a3 expression in BMMCs. Together, these data reveal selective induction of Nr4a family members in activated MCs and implicate Nr4a family nuclear receptors in the regulation of MC function.

Src homology 2 domain-containing protein B (SHB) is an adapter protein involved in the regulation of β-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 β-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 β-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.

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. [Cancer Res 2009;69(5):2141–8]

In contrast to most human autosomal genes which are expressed biallelically, the expression of imprinted genes depends on the parental origin of the allele. Prader-Willi syndrome is a neurobehavioral disorder in which the expression of active paternal alleles of imprinted genes from chromosomal region 15q11-q13 is abolished by deletions, maternal uniparental disomy or imprinting defects. We report an unusual case of maternal uniparental disomy of chromosome 15 due to a balanced translocation t(8;15)(q24.1;q21.2) leading to Prader-Willi syndrome in a 3-year-old girl.Cytogenetic investigation revealed a balanced translocation t(8;15)(q24.1;q21.2) in the patient and subsequently also in her unaffected mother. Fluorescence in situ hybridization analysis did not reveal any deletion of the PWS critical region, but methylation analysis of the SNRPN gene showed an abnormal methylation pattern indicating the absence of paternal chromosome 15. Microsatellite analysis of multiple loci and methylation-specific MLPA analysis confirmed maternal uniparental heterodisomy of chromosome 15 as the cause of PWS in the patient.This example emphasizes the importance of uniparental disomy testing in pregnancies of carriers of chromosomal aberrations with participation of chromosomes carrying imprinted genes involved in human diseases.

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.

Background Shb, 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. Results Shb 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. Conclusion Our results indicate that Shb appears to play an important modulating role on TCR signaling, thus regulating the peripheral CD4+ TH2 cell response.