Faculty Bibliography

Transforming growth factor beta (TGF-beta), a potent ubiquitous endogenous inhibitor of epithelial cell growth, is secreted as a latent molecule (LTGF-beta)requiring activation for function. TGF-beta signals through the type I(TbetaRI) and type II (TbetaRII) receptors, which cooperate to phosphorylate/activate Smad2/3, the transcriptional regulators of genes that induce cell cycle arrest. That carcinomas grow in vivo suggests that they are refractory to TGF-beta. However, this has been difficult to prove because of an inability to analyze the functional status of TGF-beta in vivo as well as lack of close physiological paradigms for carcinoma cells in vitro. The current studies demonstrate that whereas primary cultures of endometrial epithelial cells derived from normal proliferative endometrium (PE; n = 10) were dose-dependently and maximally growth inhibited by 55% +/- 5.3% with 10 pM TGF-beta1, endometrial epithelial cells derived from endometrial carcinomas (ECAs; n = 10) were unresponsive (P < or = 0.0066). To determine the mechanism of TGF-beta resistance in ECAs, we analyzed the TGF-beta signaling pathway in vivo by immunohistochemistry using specific antibodies to TbetaRI and TbetaRII, Smads, and to the phosphorylated form of Smad2 (Smad2P), an indicator of cells responding to bioactive TGF-beta. Smad2P was expressed in all of the normal endometria (n = 25), and was localized to the cytoplasm and nucleus in PE, and only nuclear in the secretory endometrium. In marked contrast, Smad2P immunostaining was weak or undetectable in ECA (n = 22; P < or = 0.001) and reduced in glandular hyperplasia (n = 25) compared with normal endometrium. However, total Smad2 and Smad7 (which inhibits Smad2 activation) levels were identical in ECA and normal tissue. Consistent with loss of downstream signaling, both TbetaRI (n = 19) and TbetaRII (n = 22) protein expression were significantly reduced in ECA compared with PE (n = 11; P < or = 0.05). By in situ hybridization, the mRNA levels of TbetaRI and TbetaRII were decreased in the carcinoma cells compared with normal PE glands, suggesting that receptor down-regulation occurs at the transcriptional level. Furthermore, a somatic frameshift mutation in the polyadenine tract at the 5' end of the TbetaR-II gene was detected in two of six cases examined. Finally, the ability of explants of ECA to activate endogenous LTGF-beta was deficient compared with normal tissue (23.5% versus 7.4%). Therefore, our results suggest that loss of Smad2 signaling in ECA may be because of down-regulation of TbetaRI and TbetaRII, and/or decreased activation of LTGF-beta. Because disruption of TGF-beta signaling occurred independent of grade or degree of invasion and was evident in premalignant hyperplasia, we conclude that inactivation of TGF-beta signaling leading to escape from normal growth control occurs at an early stage in endometrial carcinogenesis, thereby defining novel molecular targets for cancer prevention

Latent transforming growth factor beta binding protein-1 (LTBP-1) is important in regulating the localization and activation of transforming growth factor beta. In this paper is reported the isolation of the full-length Xenopus LTBP-1 cDNA from screening a neurula embryo cDNA library. Sequence analysis of XLTBP-1 cDNA revealed an open reading frame of 4518 bp encoding a 1398 amino acid protein with a molecular mass of 154.1 kDa and an isoelectric point of 4.65. The Xenopus XLTBP-1 shares 61 and 65% amino acid identity with the mouse and human LTBP-1, respectively. It contains 17 epidermal growth factor-like motifs and four eight-cysteine repeats (8-Cys). RNase protection assay revealed that XLTBP-1 is a maternal and zygotic gene, while whole-mount in situ hybridization analysis performed on embryos at different stages showed that during early Xenopus development, XLTBP-1 mRNA is expressed in the Spemann organizer, prechordal and chordal mesoderm, and later on in the organizer derived tissues. These findings suggest an important role for XLTBP-1 in embryo axis formation.

Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse

Menstrual cycle activity predisposes to ovarian epithelial tumors based on numerous epidemiological studies. We showed that the hormones involved in menstrual cycle regulation modulate cell cycle activity in these tumors in an accompanying paper. We investigated whether such hormones could also influence angiogenesis, an important determinant of tumor progression, in the same tumors. Treatment with progesterone (P4) stimulated VEGF protein secretion in 4 of 5 ovarian carcinoma cell lines examined. Northern blot analyses performed in MCV50 cells showed that this effect was accompanied by increased VEGF mRNA levels. P4 also stimulated VEGF promoter activity in these cells. Estradiol (E2) showed a similar, but substantially smaller effect on VEGF secretion which was additive to that of P4. Conditioned medium from P4-treated cells strongly stimulated angiogenesis on chicken chorio-allantoic membranes. Incubating the conditioned medium with a neutralizing anti-VEGF antibody, but not with non-specific immunoglobulins abolished this effect. Angiogenic activity was not altered by treatment of the membranes with P4 directly. We conclude that P4 can stimulate angiogenic activity via induction of VEGF secretion in some ovarian epithelial tumors. Therapeutic use of progestins may be most effective when administered in combination with an anti-angiogenic agent, at least against a subset of ovarian carcinomas

BACKGROUND: No effective long-term treatment is available for rheumatoid arthritis. Recent advances in gene therapy and cell therapy have demonstrated efficiency in collagen-induced arthritis (CIA). Interleukin-4 (IL-4) is already known to be efficient in CIA in systemic injection or administered by gene therapy. This study was designed to evaluate the effect of a non-viral gene therapy of CIA, involving injection of syngeneic fibroblasts transfected with a plasmid encoding for IL-4. METHODS: Immortalised fibroblasts from DBA/1 mice (DBA/1/0 cells) were transfected with a plasmid expressing IL-4 cDNA (DBA/1/IL-4 cells). Xenogeneic fibroblasts from Chinese hamster ovary (CHO) transfected with a plasmid expressing IL-4 cDNA (CHO/IL-4) were studied also. The cells were engrafted in mice developing CIA by subcutaneous injection of 3 x 10(6) DBA/1/0 or DBA/1/IL-4 or CHO/IL-4 cells. RESULTS: Injection of DBA/1/IL-4 cells, on days 10 and 25 after immunisation, was associated with a significant and lasting improvement in the clinical and histological evidence of joint inflammation and destruction as compared with DBA/1/0 and CHO/IL-4 cells. DBA/1/IL-4 cell treatment decreased also the production of IgG2a antibody to CII and the proliferation of CIIB-specific nodal T cells. Later treatments (engraftments on days 23 and 35 after immunisation) exerted also an anti-inflammatory effect, as evaluated on clinical and histological signs of CIA. CONCLUSIONS: Taken together, these findings indicate that systemic administration of syngeneic cells transfected with an anti-inflammatory cytokine gene, namely IL-4, with a non-viral method is effective in CIA and may attenuate the cytokine imbalance seen in this disease.

Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3(-/-)) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.

Leukocyte extravasation is initiated by interaction with endothelial selectins through selectin ligands. To understand the relative roles of E- and P-selectin in eosinophil recruitment in inflamed skin, we examined the expression of sialyl-Lewis x (sLex) structures and selectin ligands on eosinophils from patients with atopic dermatitis using whole blood flow cytometry. None of the eosinophils from the blood of patients expressed HECA452 (a lymphocyte receptor for skin homing) or CSLEX1 epitopes, and they had little avidity for soluble E-selectin. Whereas levels of the FH6 epitope (sialyl-dimeric Lex) varied on blood eosinophils, none of the infiltrative eosinophils in the skin lesions of patients expressed any type of sLex structures on the surface. In contrast, blood eosinophils bound to soluble P-selectin. The amount of P-selectin that bound to eosinophils was significantly greater in patients with atopic dermatitis than in healthy donors. PSGL-1 expression between these two groups did not differ. Furthermore, eosinophils expressed a large amount of alpha (1, 3) fucosyltransferase (FucT)-IV mRNA, but remarkably little or no FucT-VII mRNA compared with neutrophils. These data indicate that eosinophil interaction with endothelial P-selectin is far more important than interaction with E-selectin for recruitment into the inflamed skin of patients with atopic dermatitis. None of HECA452, 2H5, CSLEX1 or FH6 antigens functioned as a specific receptor to promote preferential skin infiltration via adhesion to endothelial E-selectin. FucT-IV in eosinophils may be more relavent to the generation of functional P-selectin ligand than FucT-VII.

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes

The murine p204 protein level is highest in heart and skeletal muscle. During the fusion of cultured myoblasts to myotubes, the p204 level increases due to transcription dependent on the muscle-specific MyoD protein, and p204 is phosphorylated and translocated from the nucleus to the cytoplasm. p204 overexpression accelerates myoblast fusion in differentiation medium and triggers this process even in growth medium. Here we report that p204 is required for the differentiation of C2C12 myoblasts. We propose that it enables the differentiation, at least in part, by overcoming the inhibition of the activities of the MyoD and E47 proteins by the Id proteins: Id1, Id2, and Id3. These are known to inhibit skeletal muscle differentiation by binding and blocking the activity of MyoD, E12/E47, and other myogenic basic helix-loop-helix (bHLH) proteins. Our hypothesis is based on the following findings. (i) A decrease in the p204 level in C2C12 myoblasts by antisense RNA (a) increased the level of the Id2; (b) inhibited the MyoD-, E12/E47-, and other bHLH protein-dependent accumulation of the muscle-specific myosin heavy-chain protein; and (c) inhibited the fusion of myoblasts to myotubes in differentiation medium. (ii) p204 bound to the Id proteins in vitro and in vivo. (iii) In the binding of p204 to Id2, the b segment of p204 and the HLH segment of Id2 were involved. (iv) Addition of p204 overcame the inhibition by the Id proteins of the binding of MyoD and E47 to DNA in vitro. (v) Overexpression of p204 in myoblasts (a) decreased the level of the Id proteins, even in a culture in growth medium, and (b) overcame the inhibition by the Id proteins of MyoD- and E47 dependent transcription and also overcame the inhibition by Id2 of the fusion of myoblasts to myotubes