Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family.

Using a mRNA differential screening of fibroblasts differing for the expression of c-fos we isolated a c-fos-induced growth factor (FIGF). The deduced protein sequence predicts that the cDNA codes for a new member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. Northern blot analysis shows that FIGF expression is strongly reduced in c-fos-deficient cells. Transfection of exogenous c-fos driven by a constitutive promoter restores the FIGF expression in these cells. In contrast, both PDGF and VEGF expression is unaffected by c-fos. FIGF is a secreted dimeric protein able to stimulate mitogenic activity in fibroblasts. FIGF overexpression induces morphological alterations in fibroblasts. The cells acquire a spindle-shaped morphology, become more refractive, disorganized, and detach from the plate. These results imply that FIGF is a downstream growth and morphogenic effector of c-fos. These results also suggest that the expression of FIGF in response to c-fos activation induces specific differentiation patterns and its aberrant activation contributes to the malignant phenotype of tumors.

Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.

p53-dependent cell cycle arrest induced by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts.

Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclin-dependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the G1-phase of cell cycle progression.

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase.

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.

Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tube-like structures of cultured vascular endothelial cells.

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) gene.

Using the Escherichia coli lacZ gene to identify chromosomal loci that are transcriptionally active during growth arrest of NIH 3T3 fibroblasts, we found that an mRNA expressed preferentially in serum-deprived cells specifies the previously characterized alpha-receptor (alphaR) for platelet-derived growth factor (PDGF), which mediates mitogenic responsiveness to all PDGF isoforms. Both PDGFalphaR mRNA, which was shown to include a 111-nt segment encoded by a DNA region thought to contain only intron sequences, and PDGFalphaR protein accumulated in serum-starved cells and decreased as cells resumed cycling. Elevated PDGFalphaR gene expression during serum starvation was not observed in cells that had been transformed with oncogenes erbB2, src, or raf, which prevent starvation-induced growth arrest. Our results support the view that products of certain genes expressed during growth arrest function to promote, rather than restrict, cell cycling. We suggest that accumulation of the PDGFalphaR gene product may facilitate the exiting of cells from growth arrest upon mitogenic stimulation by PDGF, leading to the state of "competence" required for cell cycling.

Aberrant platelet-derived growth factor alpha-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis.

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor.

We have been studying the role and mechanism of estrogen action in the survival and differentiation of neurons in the basal forebrain and its targets in the cerebral cortex, hippocampus, and olfactory bulb. Previous work has shown that estrogen-target neurons in these regions widely coexpress the mRNAs for the neurotrophin ligands and their receptors, suggesting a potential substrate for estrogen-neurotrophin interactions. Subsequent work indicated that estrogen regulates the expression of two neurotrophin receptor mRNAs in prototypic peripheral neural targets of nerve growth factor. We report herein that the gene encoding the neurotophin brain-derived neurotrophic factor (BDNF) contains a sequence similar to the canonical estrogen response element found in estrogen-target genes. Gel shift and DNA footprinting assays indicate that estrogen receptor-ligand complexes bind to this sequence in the BDNF gene. In vivo, BDNF mRNA was rapidly up-regulated in the cerebral cortex and the olfactory bulb of ovariectomized animals exposed to estrogen. These data suggest that estrogen may regulate BDNF transcription, supporting our hypothesis that estrogen may be in a position to influence neurotrophin-mediated cell functioning, by increasing the availability of specific neurotrophins in forebrain neurons.

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor beta 1, or platelet-derived growth factor B gene.

Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor beta 1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor beta 1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

Rescue of adult mouse motoneurons from injury-induced cell death by glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to rescue developing motoneurons in vivo and in vitro from both naturally occurring and axotomy-induced cell death. To test whether GDNF has trophic effects on adult motoneurons, we used a mouse model of injury-induced adult motoneuron degeneration. Injuring adult motoneuron axons at the exit point of the nerve from the spinal cord (avulsion) resulted in a 70% loss of motoneurons by 3 weeks following surgery and a complete loss by 6 weeks. Half of the loss was prevented by GDNF treatment. GDNF also induced an increase (hypertrophy) in the size of surviving motoneurons. These data provide strong evidence that the survival of injured adult mammalian motoneurons can be promoted by a known neurotrophic factor, suggesting the potential use of GDNF in therapeutic approaches to adult-onset motoneuron diseases such as amyotrophic lateral sclerosis.