Vitezslav Kriz

A complex role for FGF-2 in self-renewal, survival, and adhesion of human embryonic stem cells.

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor‐2 (FGF‐2), which activates FGF receptors (FGFRs) and stimulates the mitogen‐activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin‐like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF‐2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF‐2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation‐related genes and downregulation of stem cell genes. Thus, exogenous FGF‐2 reinforced the pluripotency maintenance program of intracrine FGF‐2 signaling. Consistent with this hypothesis, expression of endogenous FGF‐2 decreased during hESC differentiation and FGF‐2 knockdown‐induced hESC differentiation. In addition, FGF‐2 signaling via FGFR2 activated MAPK kinase/extracellular signal‐regulated kinase and AKT kinases, protected hESC from stress‐induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self‐renewal, cell survival, and adhesion by exogenous and endogenous FGF‐2 may synergize to maintain the undifferentiated growth of hESCs. STEM CELLS 2009;27:1847–1857

The FRK / RAK-SHB Signaling Cascade: A Versatile Signal- Transduction Pathway that Regulates Cell Survival, Differentiation and Proliferation

Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express this kinase.

Amer1/WTX couples Wnt‐induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low‐density lipoprotein receptor‐related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β‐catenin signalling, which requires Wnt‐induced formation of phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P2‐binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt‐induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P2. Amer1 translocates to the plasma membrane in a PtdIns(4,5)P2‐dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P2. Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β‐catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P2 leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.

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.

Role of tyrosine kinase signaling for beta-cell replication and survival.

Abstract Diabetes mellitus is commonly considered as a disease of a scant β-cell mass that fails to respond adequately to the functional demand. Tyrosine kinases may play a role for β-cell replication, differentiation (neoformation) and survival. Transfection of β-cells with DNA constructs coding for tyrosine kinase receptors yields a ligand-dependent increase of DNA synthesis in β-cells. A PCR-based technique was adopted to assess the repertoire of tyrosine kinases expressed in fetal islet-like structures, adult islets or RINm5F cells. Several tyrosine kinase receptors, such as the VEGFR-2 (vascular endothelial growth factor receptor 2) and c-Kit, were found to be present in pancreatic duct cells. Because ducts are thought to harbor β-cell precursor cells, these receptors may play a role for the neoformation of β-cells. The Src-like tyrosine kinase mouse Gtk (previously named Bsk/Iyk) is expressed in islet cells, and was found to inhibit cell proliferation. Furthermore, it conferred decreased viability in response to cytokine exposure. Shb is a Src homology 2 domain adaptor protein which participates in tyrosine kinase signaling. Transgenic mice overexpressing Shb in β-cells exhibit an increase in the neonatal β-cell mass, an improved glucose homeostasis, but also decreased survival in response to cytokines and streptozotocin. It is concluded that tyrosine kinase signaling may generate multiple responses in β-cells, involving proliferation, survival and differentiation.

Structural and functional characterization of the Wnt inhibitor APC membrane recruitment 1 (Amer1)

Amer1/WTX binds to the tumor suppressor adenomatous polyposis coli and acts as an inhibitor of Wnt signaling by inducing β-catenin degradation. We show here that Amer1 directly interacts with the armadillo repeats of β-catenin via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and that Amer1 assembles the β-catenin destruction complex at the plasma membrane by recruiting β-catenin, adenomatous polyposis coli, and Axin/Conductin. Deletion or specific mutations of the membrane binding domain of Amer1 abolish its membrane localization and abrogate negative control of Wnt signaling, which can be restored by artificial targeting of Amer1 to the plasma membrane. In line, a natural splice variant of Amer1 lacking the plasma membrane localization domain is deficient for Wnt inhibition. Knockdown of Amer1 leads to the activation of Wnt target genes, preferentially in dense compared with sparse cell cultures, suggesting that Amer1 function is regulated by cell contacts. Amer1 stabilizes Axin and counteracts Wnt-induced degradation of Axin, which requires membrane localization of Amer1. The data suggest that Amer1 exerts its negative regulatory role in Wnt signaling by acting as a scaffold protein for the β-catenin destruction complex and promoting stabilization of Axin at the plasma membrane.

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 SHB adapter protein is required for efficient multilineage differentiation of mouse embryonic stem cells

The SH2 domain-containing adapter protein SHB transmits signals from receptor tyrosine kinases regulating diverse processes such as apoptosis and differentiation. To elucidate a role for SHB in cell differentiation, wild-type and R522K (inactive SH2 domain-mutant) SHB were transfected and expressed in mouse embryonic stem (ES) cells. Microarray analysis using Affymetrix U74A chips on undifferentiated ES cells and expression of selected differentiation markers after generation of embryoid bodies were subsequently assessed. Wild-type SHB altered the expression of 16 genes in undifferentiated ES cells, many of which have been found to relate to neural cell function. R522K-SHB altered the expression of 128 genes in undifferentiated ES cells, the majority of which were decreased, including several transcription factors related to development. When grown as embryoid bodies, after 4 days R522K-SHB ES cells were already found to display a different morphological appearance, with an impaired cavity formation that occurred in the absence of altered OCT4 expression. This impairment was reversed by exogenous addition of Matrigel. In addition, R522K-SHB embryoid bodies displayed reduced mRNA contents of the liver protein albumin, the pancreatic proteins amylase, glucagon and insulin after 20 days of differentiation. Matrigel did not restore the impaired expression of albumin in the R522K-SHB cells. Expression of the mesodermal marker cardiac actin and the neural marker neurofilament heavy chain alpha was not affected by wild-type or R522K-SHB overexpression. It is concluded that SHB is required for efficient differentiation of ES cells into embryoid bodies with normal cavities and cells belonging to endodermal lineages.

The SHB Adapter Protein Is Required for Normal Maturation of Mesoderm during in Vitro Differentiation of Embryonic Stem Cells

Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in β-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.