Mice lacking the homologue of the human 22q11.2 gene CRKL phenocopy neurocristopathies of DiGeorge syndrome.

Heterozygous deletions within human chromosome 22q11 are the genetic basis of DiGeorge/velocardiofacial syndrome (DGS/VCFS), the most common deletion syndrome (1 in 4,000 live births) in humans. CRKL maps within the common deletion region for DGS/VCFS (ref. 2) and encodes an SH2-SH3-SH3 adapter protein closely related to the Crk gene products. Here we report that mice homozygous for a targeted null mutation at the CrkL locus (gene symbol Crkol for mice) exhibit defects in multiple cranial and cardiac neural crest derivatives including the cranial ganglia, aortic arch arteries, cardiac outflow tract, thymus, parathyroid glands and craniofacial structures. We show that the migration and early expansion of neural crest cells is unaffected in Crkol-/- embryos. These results therefore indicate an essential stage- and tissue-specific role for Crkol in the function, differentiation, and/or survival of neural crest cells during development. The similarity between the Crkol-/- phenotype and the clinical manifestations of DGS/VCFS implicate defects in CRKL-mediated signaling pathways as part of the molecular mechanism underlying this syndrome.

Colony-stimulating factor-1 receptor utilizes multiple signaling pathways to induce cyclin D2 expression.

Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src(-/-) bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes.

Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases.

SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, an Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins such as fibronectin and laminin also induced the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses were markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase did not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibited activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 did not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix.

Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells.

The ROSA beta geo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral beta geo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5' ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans.

Activation of the epidermal growth factor receptor by skin tumor promoters and in skin tumors from SENCAR mice.

The present study was designed to further investigate the role of the epidermal growth factor receptor (EGFr) in mouse skin tumor promotion by evaluating the status of the EGFr in tumor promoter-treated mouse epidermis and in mouse skin tumors. Female SENCAR mice received three topical treatments of either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or the nonphorbol esters okadaic acid and chrysarobin. Membrane proteins from SENCAR mouse epidermis were isolated 6 h after the last treatment, and the phosphotyrosine content of the EGFr and several potential substrates were examined by Western blot analysis. The results indicated that multiple applications of all three tumor promoters led to an increase in the phosphotyrosine content of the EGFr and also of several lower molecular weight proteins (M(r) approximately 80,000-85,000). Phosphorylation of PLC gamma 1 on tyrosine residues could not be detected in tumor promoter-treated mouse epidermis when the phosphotyrosine content of the EGFr was elevated or in cultured keratinocytes exposed to exogenous EGF. When two tyrosine kinase inhibitors (tyrphostins RG50864 and RG13022) were incorporated into the treatment regimens, the TPA-induced epidermal hyperplasia and cell proliferation were effectively blocked, and the TPA-stimulated EGFr tyrosine phosphorylation was significantly reduced. Examination of the phosphotyrosine content of epidermal membrane proteins isolated from skin papillomas revealed that the EGFr also had elevated phosphotyrosine levels. These results demonstrate that multiple topical treatments with both phorbol ester and nonphorbol ester tumor promoters lead to activation of the EGFr tyrosine kinase in mouse epidermis. In addition, these data suggest that signaling through the EGFr pathway plays an important role in the tumor promotion stage of multistage carcinogenesis in mouse skin.

Specific and redundant roles of Src and Fyn in organizing the cytoskeleton.

Mouse embryos lacking Csk, a negative regulator of Src family kinases, exhibit defects in neurulation and die at mid-gestation. To determine the role of activated Src family kinases in the csk- phenotype, we have introduced mutations in the src and fyn genes into the csk- mutant background. Genetic analysis reveals that src, but not fyn, is partly epistatic to the csk gene. Biochemical analysis indicates that several cytoskeletal proteins are hyperphosphorylated on tyrosine residues in csk- cells. Regulation of cortactin and tensin hyperphosphorylation is Src-dependent, whereas focal adhesion kinase and paxillin hyperphosphorylation is partly dependent on both Src and Fyn. Furthermore, the src- mutation can restore the normal distribution of cortactin and partly correct filamentous actin organization in csk-cells. Thus, Src family kinases have both specific and overlapping functions in regulation of the cytoskeleton. The disturbance of these functions may be a molecular basis for the phenotype exhibited by csk- mutants.

Genetics of signal transduction: tales from the mouse.

Recent progress in understanding signal transduction owes much to new genetic approaches, first by unraveling the molecular basis of classic mutations, and then by the use of gene targeting. Recent studies have examined mammalian signal transduction from cell surface to nucleus, especially ligand-receptor systems and cytosolic signal transducers.

Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice.

All Src family non-receptor tyrosine kinases are negatively regulated by phosphorylation at a carboxy-terminal tyrosine. To analyze the significance of this regulation during development, we have generated mice deficient in Csk, a kinase that phosphorylates this tyrosine, by gene targeting in embryonic stem cells. Homozygous mutant embryos exhibit a complex phenotype that includes defects in the neural tube and die between day 9 and day 10 of gestation. Cells derived from these embryos exhibit an order of magnitude increase in activity of Src and the related Fyn kinase. Phosphorylation at the carboxy-terminal tyrosine of Src was reduced but not eliminated and was accompanied by increased phosphorylation at another key tyrosine residue. These results demonstrate that Src family kinase activity is critically dependent on phosphorylation by Csk and suggest that the regulation of kinase activity may be essential during embryogenesis.

Comparison of 12-O-tetradecanoylphorbol-13-acetate and teleocidin for induction of epidermal hyperplasia, activation of epidermal PKC isozymes and skin tumor promotion in SENCAR and C57BL/6 mice.

The present study compared the ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin to induce sustained epidermal hyperplasia, activate partially purified epidermal protein kinase C (PKC) isozymes and promote skin tumors in SENCAR and C57BL/6 mice. Teleocidin was less effective than TPA on a molar basis for inducing sustained epidermal hyperplasia, promoting skin tumors and activating partially purified epidermal PKC isozymes in vitro when examined using SENCAR mice. In contrast, teleocidin was more effective than TPA on a molar basis for inducing sustained epidermal hyperplasia, approximately equi-effective for promoting skin tumors and significantly less effective for activating PKC isozymes in vitro when examined using C57BL/6 mice. Despite the differences in response of C57BL/6 mice to TPA and teleocidin, this mouse strain was still highly resistant to skin tumor promotion by both types of promoters when compared with SENCAR mice. The current results, when considered in light of our recent studies (Cancer Res., 51, 1398-1405, 1991), indicate that C57BL/6 are generally resistant to a variety of classes of skin tumor promoters, including the teleocidins. In addition, except for the phorbol esters, the induction of sustained epidermal hyperplasia does not appear to be as good a marker for overall promotion responsiveness between SENCAR and C57BL/6 mice with other classes of tumor promoters; although the induction of a significant sustained hyperplasia in the latter mouse strain did yield a weak tumor response. Taken together, the current data suggest that factors in addition to the induction of sustained epidermal hyperplasia, control responsiveness of C57BL/6 mice to skin tumor promotion by diverse promoting stimuli.

Further genetic analyses of skin tumor promoter susceptibility using inbred and recombinant inbred mice.

To explore further the genetics of susceptibility to skin tumor promotion in inbred mice, several aspects of responsiveness to 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined in C3H/He mice and segregating crosses between this mouse strain and C57BL/6 mice as well as BXD and BXH recombinant inbred (RI) strains. Dose-response relationships were established for skin tumor promotion by TPA following initiation with 7,12-dimethylbenz[a]anthracene in C3H/He and B6C3F1, as well as several other mouse stocks and strains included for comparison. The relative responsiveness to TPA skin tumor promotion was: SENCAR much greater than DBA/2 greater than C3H/He approximately B6D2F1 greater than B6C3F1 much greater than C57BL/6. Analyses of the susceptibility of B6C3F2 and B6C3F1 x C57BL/6 backcross mice suggested that a minimum of two dominant genetic loci control responsiveness to phorbol ester promotion in these mice. Further analysis of BXH and BXD RI strains suggested the presence of four distinct promotion-responsive phenotypes controlled by a minimum of two genetic loci. The existence of a 'hyper-responsive' phenotype in the sets of RI strains, however, suggests that a third, recessive locus also may play a role in controlling responsiveness to TPA promotion. At 48 h after the last of four applications of TPA, marked hyperplasia and an increase in dark basal keratinocytes were observed in C3H/He mice, whereas in B6C3F1 mice the response in these parameters was intermediate between C3H/He and C57BL/6 mice. A marked dermal inflammation, as determined by infiltration of polymorphonuclear cells, was observed in C3H/He and B6C3F1 mice, whereas little was noted in C57BL/6 mice. Furthermore, histological evaluations of selected BXD RI strains revealed a significant correlation between the magnitude of the hyperplasia response and the percentage of mice bearing tumors. The present data, in conjunction with our previous studies, confirm that the major gene(s) controlling susceptibility to tumor promoter induced by TPA in two sensitive strains (i.e. DBA/2 C3H/He) are similar or closely linked to those for induction of sustained hyperplasia. In addition, the present data provide new evidence for a model where allelic differences at a minimum of three loci contribute to gene differences in susceptibility to phorbol ester promotion DBA/2 and C3H/He versus C57BL/6 mice.

Enhanced induction of epidermal ornithine decarboxylase activity in C57BL/6 compared to DBA/2 mice by protein kinase C-activating skin tumor promoters: relevance to genetically mediated differences in promotion susceptibility.

Previous work from our laboratory demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) or a synthetic diacylglycerol induced significantly higher epidermal ornithine decarboxylase (ODC) activity in C57BL/6 than in DBA/2 mice. To understand further the genetic basis for this strain difference, two tumor promoters were evaluated for their effects on epidermal ODC activity: teleocidin, which activates protein kinase C (PKC); and 1,8-dihydroxyl-3-methyl-9-anthrone (chrysarobin), which does not. In addition, the ODC induction response in B6D2F1 offspring and BXD recombinant inbred (RI) strains was examined following multiple treatments with TPA. A single topical application of teleocidin to mouse dorsal skin led to the hyperinduction of epidermal ODC activity in C57BL/6 mice. In contrast, while chrysarobin induced epidermal ODC activity, no significant differences in the magnitude of this response were observed in SENCAR, DBA/2 or C57BL/6 mice. Consistent with our previous findings, the magnitude of ODC induction by teleocidin in these three mouse lines (C57BL/6 greater than SENCAR greater than DBA/2) did not correlate with their susceptibility to tumor promotion by TPA (SENCAR greater than DBA/2 greater than C57BL/6). ODC activity induced by multiple application of TPA in B6DF1 mice, whose susceptibility to phorbol ester tumor promotion is inherited as an incomplete dominant trait, was comparable to that induced in C57BL/6 mice at all the doses examined. Cluster analysis of TPA-induced ODC activity in BXD RI strains allowed us tentatively to group them into four or five phenotypes and to estimate a minimum of two genetic loci controlling TPA-induced ODC activity. Furthermore, in BXD RI strains, there was no apparent relationship between the magnitude of ODC induction and responsiveness to tumor promotion or sustained hyperplasia. Collectively, these results suggest that hyperinducibility of ODC in response to PKC-activating tumor promoters is inherited as an autosomal dominant trait, and that genetic determinants for ODC induction, at least in C57BL/6 and DBA/2 mice, appear completely independent of those controlling tumor promotion susceptibility.

Evidence for autocrine/paracrine growth stimulation by transforming growth factor-alpha during the process of skin tumor promotion.

A single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin decreased 125I-labeled epidermal growth factor (EGF) binding in epidermal membrane preparations within 1 h while 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) gradually reduced binding with maximum inhibition at 15 h. Subsequently, 125I-EGF binding increased to approximately 200% of control in epidermal membrane preparations from both TPA- and chrysarobin-treated mice. A single application of TPA but not chrysarobin resulted in a rapid translocation of protein kinase C (PKC) to the membrane; however, treatment with both promoters ultimately led to a time-dependent loss of PKC activity in both membrane and cytosol fractions. The initial inhibition of 125I-EGF binding was sustained for at least 24 h after single and multiple treatments with both promoting agents. Acid washing restored EGF binding to control levels in membrane preparations obtained 24 h after a single application, whereas acid washing of membrane preparations obtained 24 h after a second application of TPA or chrysarobin increased binding (2.5-fold and 1.5-fold that of the control, respectively). The presence of increased amounts of ligands for the EGF receptor in tumor promoter-treated epidermis was initially confirmed in 125I-EGF binding competition experiments using NRK-49F cells. A single topical application of TPA or chrysarobin induced elevated levels of transforming growth factor-alpha (TGF-alpha) mRNA at 6 h or 15-24 h, respectively. Elevated levels of a TGF-alpha precursor (21 kDa) were subsequently observed in cytosol and membrane preparations after single and multiple applications of TPA or chrysarobin. These results suggest that repeated topical application of tumor promoters may lead to sustained loss of a negative-feedback mechanism involving phosphorylation at Thr-654 of the EGF receptor by PKC. The concomitant elevation of ligands, such as TGF-alpha, may provide a mechanism for sustained cell proliferation essential for skin tumor promotion.

Differential mechanism for the inhibition of epidermal growth factor binding to its receptor on mouse keratinocytes by anthrones and phorbol esters.

1,8-Dihydroxy-3-methyl-9-anthrone (chrysarobin), a potent anthrone tumor promoter, reduced [125I] epidermal growth factor (EGF) binding to its receptor in primary epidermal cells from SENCAR mice maintained in low Ca2+ containing medium. The time course for this effect with chrysarobin was different from that of 12-O-tetradecanoylphorbol-13-acetate (TPA). Maximum inhibition of [125I]EGF binding was observed at 18 h versus 1 h respectively. Scatchard analyses revealed that the inhibition by chrysarobin was due to a decrease in the number of both high- and low-affinity classes of EGF receptors. In contrast, TPA caused a rapid inhibition of EGF binding, primarily due to a loss of high-affinity receptors. The mechanism by which chrysarobin inhibited the binding of EGF to its receptor involved neither direct activation nor membrane translocation of epidermal protein kinase C, whereas the rapid decrease in EGF binding induced by TPA was consistent with its ability to activate protein kinase C. Structure-activity relationships for EGF binding inhibition by anthrones revealed that inhibition was inversely proportional to chain length at the C10-position, which correlated closely with oxidation rate and skin tumor-promoting activity. alpha-Tocopherol was able to block partially the effect of chrysarobin but not TPA on EGF binding. These results suggest that oxidation at position C10 is at least partially responsible for the inhibition of EGF binding induced by chrysarobin. Furthermore, these studies support the hypothesis that changes in EGF receptor binding and/or function may play a role in skin tumor promotion by diverse classes of promoting agents.