Identification of an angiogenic mitogen selective for endocrine gland endothelium.

The known endothelial mitogens stimulate growth of vascular endothelial cells without regard to their tissue of origin. Here we report a growth factor that is expressed largely in one type of tissue and acts selectively on one type of endothelium. This molecule, called endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), induced proliferation, migration and fenestration (the formation of membrane discontinuities) in capillary endothelial cells derived from endocrine glands. However, EG-VEGF had little or no effect on a variety of other endothelial and non-endothelial cell types tested. Similar to VEGF, EG-VEGF possesses a HIF-1 binding site, and its expression is induced by hypoxia. Both EG-VEGF and VEGF resulted in extensive angiogenesis and cyst formation when delivered in the ovary. However, unlike VEGF, EG-VEGF failed to promote angiogenesis in the cornea or skeletal muscle. Expression of human EG-VEGF messenger RNA is restricted to the steroidogenic glands, ovary, testis, adrenal and placenta and is often complementary to the expression of VEGF, suggesting that these molecules function in a coordinated manner. EG-VEGF is an example of a class of highly specific mitogens that act to regulate proliferation and differentiation of the vascular endothelium in a tissue-specific manner.

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants.

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.

Strain-dependent myeloid hyperplasia, growth deficiency, and accelerated cell cycle in mice lacking the Rb-related p107 gene.

To investigate the function of the Rb-related p107 gene, a null mutation in p107 was introduced into the germ line of mice and bred into a BALB/cJ genetic background. Mice lacking p107 were viable and fertile but displayed impaired growth, reaching about 50% of normal weight by 21 days of age. Mutant mice exhibited a diathetic myeloproliferative disorder characterized by ectopic myeloid hyperplasia in the spleen and liver. Embryonic p107(-/-) fibroblasts and primary myoblasts isolated from adult p107(-/-) mice displayed a striking twofold acceleration in doubling time. However, cell sort analysis indicated that the fraction of cells in G1, S, and G2 was unaltered, suggesting that the different phases of the cell cycle in p107(-/-) cells was uniformly reduced by a factor of 2. Western analysis of cyclin expression in synchronized p107(-/-) fibroblasts revealed that expression of cyclins E and A preceded that of D1. Mutant embryos expressed approximately twice the normal level of Rb, whereas p130 levels were unaltered. Lastly, mutant mice reverted to a wild-type phenotype following a single backcross with C57BL/6J mice, suggesting the existence of modifier genes that have potentially epistatic relationships with p107. Therefore, we conclude that p107 is an important player in negatively regulating the rate of progression of the cell cycle, but in a strain-dependent manner.

Strain-dependent embryonic lethality in mice lacking the retinoblastoma-related p130 gene.

The retinoblastoma-related p130 protein is a member of a conserved family, consisting of Rb, p107 and p130, which are believed to play important roles in cell-cycle control and cellular differentiation. We have generated a null mutation in p130 by gene targeting and crossed the null allele into Balb/cJ and C57BL/6J strains of mice. In an enriched Balb/cJ genetic background, p130(-/-) embryos displayed arrested growth and died between embryonic days 11 and 13. Histological analysis revealed varying degrees of disorganization in neural and dermamyotomal structures. Immunohistochemistry with antibody reactive with Islet-1 indicated markedly reduced numbers of neurons in the spinal cord and dorsal root ganglia. Immunohistochemistry with antibody reactive with desmin indicated a similar reduction in the number of differentiated myocytes in the myotome. The myocardium of mutant embryos was abnormally thin and resembled an earlier staged two-chambered heart consisting of the bulbus cordis and the ventricular chamber. TUNEL analysis indicated the presence of extensive apoptosis in various tissues including the neural tube, the brain, the dermomyotome, but not the heart. Immunohistochemistry with antibody reactive with PCNA revealed increased cellular proliferation in the neural tube and the brain, and decreased proliferation in the heart. The placentas of p130(-/-) embryos did not display elevated apoptosis and were indistinguishable from wild type suggesting that the phenotype was not due to placental failure. Following a single cross with the C57BL/6 mice, p130(-/-) animals were derived that were viable and fertile. These results indicate that p130 in a Balb/cJ genetic background plays an essential role that is required for normal development. Moreover, our experiments establish that second-site modifier genes exist that have an epistatic relationship with p130.

Detection of intracellular calcium elevations in Xenopus laevis oocytes: aequorin luminescence versus electrophysiology.

Detection of receptor expression in Xenopus oocytes often relies upon functional coupling to second messengers such as Ca2+ or cyclic adenosine monophosphate. To detect intracellular Ca2+, electrophysiological measurement of the endogenous Ca(2+)-activated chloride current (ICl(Ca)) is often used (Dascal, 1987). An alternative utilizes the Ca2+ sensing, bioluminescent protein aequorin (Parker and Miledi(1986) Proc. R. Soc. Lond. B, 228: 307-315; Giladi and Spindel (1991) BioTechniques, 10: 744-747). In the present study the sensitivities of aequorin and electrophysiology for detecting receptor-mediated Ca2+ transients were compared. Assays were performed on the same batches of oocytes using either animal serum or ligands of exogenous receptors to generate inositol 1,4,5-trisphosphate (InsP3) and ultimately elevate intracellular Ca2+. Signal amplitudes were controlled by titrating the concentration of animal serum, or titrating the amount of receptor mRNA injected. Both assays detected signals with high concentrations of animal serum, or with high receptor density. However, aequorin signals were not detected in experiments with average ICl(Ca) current amplitudes below 200 nA. To further evaluate the differences between these two techniques, membrane current and bioluminescence were measured simultaneously. Results of these studies suggest that the signals differ due to the spatial distribution of aequorin, the chloride channels, and the calcium release sites.

Cloning and expression of the Rb-related mouse p130 mRNA.

The mouse p130 cDNA was cloned from a thymus-derived library using the human p130 cDNA as a probe. The 4515-bp mouse cDNA encodes a putative 1092 aa protein with predicted molecular mass of 123 kD. Comparison of mouse and human sequences reveals the mouse protein lacks 43 aa in a conserved domain, relative to the human sequence, located amino-terminal to the pocket region. Northern analysis of P19 embryonal carcinoma (EC) and RA-induced P19 cultures indicates p130 mRNA is not expressed at detectable levels in undifferentiated stem cells and is strongly upregulated after post-mitotic neurons begin to accumulate. Northern analysis of adult mouse tissues indicated that the 4.8 kb p130 mRNA is expressed ubiquitously, however, a putative 5'-truncated 1.7 kb isoform is detected solely in testis. Forced expression of mouse p130 induced growth suppression of P19 EC cells and Western analysis of transfected P19 cells suggested the cloned cDNA encodes the full-length p130 protein.

bFGF and LIF signaling activates STAT3 in proliferating myoblasts.

Different mitogens elicit similar effects on growth and differentiation of skeletal muscle, suggesting that potential overlap exists in the signaling cascades activated by such factors. To investigate this possibility, we examined the status of STAT and ERK proteins in C2C12 myoblasts and myotubes following stimulation with bFGF or LIF. Both STAT1 and STAT3 as well as ERK1 and ERK2 proteins were detectable in extracts of myoblasts. LIF stimulation of myoblasts lead to rapid phosphorylation on tyrosine of STAT3 and of ERKs 1 and 2. Similarly, bFGF stimulation of myoblasts resulted in the tyrosine phosphorylation of STAT3. However, unlike LIF, the bFGF induced tyrosine phosphorylation of STAT3 appeared cyclical, with recurrent peaks of phosphorylation even after prolonged exposure. By contrast, STAT1 remained unphosphorylated in myoblasts treated with bFGF or LIF. In differentiated myotubes, LIF treatment resulted in the tyrosine phosphorylation of both STAT3 and STAT1, but ERK phosphorylation was not detectable, and bFGF treatment did not lead to STAT1 or STAT3 tyrosine phosphorylation. Therefore these observations suggest that disparate mitogens car activate similar downstream effectors in proliferating myoblasts.