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J Capdevila

Publications and source records attributed to J Capdevila.

At least 19 recordsLinked to original sources
PubMed2001

WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo.

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.

Animals↗
PubMed2001

Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis.

The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosynthesis, their cellular and/or organ physiological functions remain to be fully characterized. Here we show that the cytochrome P450 epoxygenase arachidonic acid metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET) inhibits apoptosis induced by serum withdrawal, H(2)O(2), etoposide, or excess free arachidonic acid (AA), as determined by DNA laddering, Hoechst staining, and fluorescein isothiocyanate-labeled annexin V binding. In the stable transfectants (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide production within 1 h and elevated ceramide levels for up to 48 h, the longest time tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid restored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide markedly increased apoptosis in quiescent Vector cells as well as BM3 cells, and apoptosis was prevented by pretreatment of Vector cells with exogenous 14,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase inhibitor fumonisin B1 did not affect AA-induced ceramide production and apoptosis; in contrast, these effects of AA were blocked by the neutral sphingomyelinase inhibitor scyphostatin. The pan-caspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosis. The antiapoptotic effects of 14,15-EET were blocked by two mechanistically and structurally distinct phosphatidylinositol-3 (PI-3) kinase inhibitors, wortmannin and LY294002, but not by the specific mitogen-activated protein kinase kinase inhibitor PD98059. Immunoprecipitation followed by an in vitro kinase assay revealed activation of Akt kinase within 10 min after 14,15-EET addition, which was completely abolished by either wortmannin or LY294002 pretreatment. In summary, the present studies demonstrated that 14,15-EET inhibits apoptosis by activation of a PI-3 kinase-Akt signaling pathway. Furthermore, cytochrome P450 epoxygenase promotes cell survival both by production of 14,15-EET and by metabolism of unesterified AA, thereby preventing activation of the neutral sphingomyelinase pathway and proapoptotic ceramide formation.

Animals↗
PubMed2001

Patterning mechanisms controlling vertebrate limb development.

Vertebrate limb buds are embryonic structures for which much molecular and cellular data are known regarding the mechanisms that control pattern formation during development. Specialized regions of the developing limb bud, such as the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the non-ridge ectoderm, direct and coordinate the development of the limb bud along the anterior-posterior (AP), dorsal-ventral (DV), and proximal-distal (PD) axes, giving rise to a stereotyped pattern of elements well conserved among tetrapods. In recent years, specific gene functions have been shown to mediate the organizing and patterning activities of the ZPA, the AER, and the non-ridge ectoderm. The analysis of these gene functions has revealed the existence of complex interactions between signaling pathways operated by secreted factors of the HH, TGF-beta/BMP, WNT, and FGF superfamilies, which interact with many other genetic networks to control limb positioning, outgrowth, and patterning. The study of limb development has helped to establish paradigms for the analysis of pattern formation in many other embryonic structures and organs.

Animals↗
PubMed2001

Wnt signaling and PKA control Nodal expression and left-right determination in the chick embryo.

Expression of the Nodal gene, which encodes a member of the TGFbeta superfamily of secreted factors, localizes to the left side of the developing embryo in all vertebrates examined so far. This asymmetric pattern correlates with normal development of the left-right axis. We now show that the Wnt and PKA signaling pathways control left-right determination in the chick embryo through Nodal. A Wnt/beta-catenin pathway controls Nodal expression in and around Hensen's node, without affecting the upstream regulators Sonic hedgehog, Car and Fibroblast Growth Factor 8. Transcription of Nodal is also positively regulated by a protein kinase A-dependent pathway. Both the adhesion protein N-cadherin and PKI (an endogenous protein kinase A inhibitor) are localized to the right side of the node and may contribute to restrict Nodal activation by Wnt signaling and PKA to the left side of the node.

Alkaline Phosphatase↗
PubMed2000

Perspectives on the evolutionary origin of tetrapod limbs.

The study of the origin and evolution of the tetrapod limb has benefited enormously from the confluence of molecular and paleontological data. In the last two decades, our knowledge of the basic molecular mechanisms that control limb development has grown exponentially, and developmental biologists now have the possibility of combining molecular data with many available descriptions of the fossil record of vertebrate fins and limbs. This synthesis of developmental and evolutionary biology has the potential to unveil the sequence of molecular changes that culminated in the adoption of the basic tetrapod limb plan.

Animals↗
PubMed2000

Overexpression of C-terminal Src kinase blocks 14, 15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis.

We have previously reported that 14,15-epoxyeicosatrienoic acid (14, 15-EET) is a potent mitogen for the renal epithelial cell line, LLCPKcl4. This mitogenic effect is dependent upon activation of a protein-tyrosine kinase cascade that results in activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Because of suggestive evidence that 14,15-EET also activated Src in these cells, we stably transfected LLCPKcl4 with an expression construct of the C-terminal Src kinase (CSK), which inhibits Src family kinase activity. In vitro Src kinase activity assays confirmed that in empty vector-transfected cells (Vector cells), 14, 15-EET increased Src kinase activity, while in clones overexpressing CSK mRNA and immunoreactive protein (CSK cells), 14,15-EET-induced activation of Src was almost completely blocked (94% inhibition). Of interest, epidermal growth factor (EGF) and fetal bovine serum (FBS) also increased Src activity in Vector cells, but not in CSK cells, further confirming the ability of CSK overexpression to prevent Src activation. CSK cells failed to increase [(3)H]thymidine incorporation in response to exogenous 14,15-EET. In contrast, both EGF and FBS significantly increased [(3)H]thymidine incorporation in CSK cells. Immunoprecipitation with anti-phosphotyrosine antibodies and immunoblotting with an antibody against extracellular signal-regulated kinase (ERK) indicated that in CSK cells, 14,15-EET failed to activate ERK1 and ERK2; however, EGF- and FBS-induced activation of ERKs was not different from that seen in Vector cells. In Vector cells, the 14,15-EET-stimulated tyrosine phosphorylation of ERKs was blocked by pretreatment with 1 microm PP2, a selective inhibitor of Src kinases. The present study demonstrates that 14, 15-EET exerts its mitogenic effects predominantly through a Src kinase-mediated pathway, which is the most upstream signaling step determined to date in the 14,15-EET-activated tyrosine kinase cascade in renal epithelial cells.

8,11,14-Eicosatrienoic Acid↗
PubMed2000

Glycine(B) receptor antagonists and partial agonists prevent memory deficits in inhibitory avoidance learning.

Activation of N-methyl-d-aspartate (NMDA) receptors has been hypothesized to mediate certain forms of learning and memory. This hypothesis is based on the ability of competitive and uncompetitive NMDA receptor antagonists to disrupt learning. We investigated the effects of glycine site antagonists and partial agonists on deficits of acquisition (learning) and consolidation (memory) in a single trial inhibitory avoidance learning paradigm. Posttraining administration of either hypoxia (exposure to 7% oxygen) or the convulsant drug pentylenetetrazole (PTZ) (45 mg/kg) to mice impaired consolidation without producing neuronal cell death. Pretreatment with the competitive glycine antagonist 7-chlorokynurenic acid (7KYN) and the glycine partial agonists 1-aminocyclopropanecarboxylic acid (ACPC) and (+)HA-966 prevented memory deficits induced by hypoxia and PTZ, but did not affect scopolamine-induced learning impairment. In addition, ACPC prevented consolidation deficits evoked by a nonexcitotoxic concentration of l-trans-pyrrolidine-2, 4-dicarboxylate, a competitive inhibitor of glutamate transport that increases extracellular levels of glutamate. Moreover, (+)HA-966, 7KYN, and ACPC facilitated both acquisition and consolidation of inhibitory avoidance training, an effect that was dose-dependent and reversed by glycine. These results indicate that memory deficits induced by both hypoxia and PTZ involve NMDA receptor activation. Furthermore, the present findings demonstrate that glycine site antagonists and partial agonists prevent memory deficits of inhibitory avoidance learning by affecting consolidation, but not acquisition processes.

Animals↗
PubMed2000

Hedgehog signaling in vertebrate and invertebrate limb patterning.

Invertebrate and vertebrate limbs have very different anatomies and modes of development. Despite these differences, recent studies demonstrate that a significant overlap exists in the signals used to pattern invertebrate and vertebrate limbs. One of these signaling molecules is Hedgehog, a secreted protein that functions to coordinate growth and proliferation along the anterior-posterior axis of developing limbs. Recent studies indicate that the mechanism of action, regulation and function of Hedgehog signaling in Drosophila and vertebrate limb development are often quite similar, yet at other times are distinct. Here we highlight the similarities and differences between the use of Hedgehog signaling in these two systems.

Animals↗
PubMed1999

Multiple left-right asymmetry defects in Shh(-/-) mutant mice unveil a convergence of the shh and retinoic acid pathways in the control of Lefty-1.

Asymmetric expression of Sonic hedgehog (Shh) in Hensen's node of the chicken embryo plays a key role in the genetic cascade that controls left-right asymmetry, but its involvement in left-right specification in other vertebrates remains unclear. We show that mouse embryos lacking Shh display a variety of laterality defects, including pulmonary left isomerism, alterations of heart looping, and randomization of axial turning. Expression of the left-specific gene Lefty-1 is absent in Shh(-/-) embryos, suggesting that the observed laterality defects could be the result of the lack of Lefty-1. We also demonstrate that retinoic acid (RA) controls Lefty-1 expression in a pathway downstream or parallel to Shh. Further, we provide evidence that RA controls left-right development across vertebrate species. Thus, the roles of Shh and RA in left-right specification indeed are conserved among vertebrates, and the Shh and RA pathways converge in the control of Lefty-1.

Animals↗
PubMed1999

The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.

In the chick embryo, left-right asymmetric patterns of gene expression in the lateral plate mesoderm are initiated by signals located in and around Hensen's node. Here we show that Caronte (Car), a secreted protein encoded by a member of the Cerberus/Dan gene family, mediates the Sonic hedgehog (Shh)-dependent induction of left-specific genes in the lateral plate mesoderm. Car is induced by Shh and repressed by fibroblast growth factor-8 (FGF-8). Car activates the expression of Nodal by antagonizing a repressive activity of bone morphogenic proteins (BMPs). Our results define a complex network of antagonistic molecular interactions between Activin, FGF-8, Lefty-1, Nodal, BMPs and Car that cooperate to control left-right asymmetry in the chick embryo.

Amino Acid Sequence↗
PubMed1999

Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor.

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio- and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241. 82 ng/10(8) cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PLA2-AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.

8,11,14-Eicosatrienoic Acid↗
PubMed1999

Extracellular modulation of the Hedgehog, Wnt and TGF-beta signalling pathways during embryonic development.

Localized embryonic expression of members of the Hedgehog, Wnt and TGF-beta families of secreted factors has been shown to organize pattern and provide positional information in many developing systems. Recently, several extracellular factors have been described that act either as facilitators or inhibitors of the activities of those secreted proteins. The variety of molecular strategies involved in the extracellular modulation of signalling activities in the embryo underscores the importance of maintaining a tight spatial and temporal control of the activities of organizing centers during development.

Animals↗
PubMed1999

Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin.

The mechanisms controlling growth and patterning along the proximal-distal axis of the vertebrate limb are yet to be understood. We show that restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb development, since ectopic Meis2 severely disrupts limb outgrowth. We also uncover an antagonistic relationship between the secreted factors Gremlin and BMPs required to maintain the Shh/FGF loop that regulates distal outgrowth. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 expression to the proximal limb bud. Moreover, combinations of BMPs and AER factors are sufficient to distalize proximal limb cells. Our results unveil a novel set of proximal-distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb.

Animals↗
PubMed1998

Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells.

In our present studies utilizing a well characterized proximal tubule cell line, LLCPKcl4, we determined that all four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) stimulated [3H]thymidine incorporation, with 14,15-EET being the most potent. In contrast, no mitogenic effects were seen with arachidonic acid, other cP450 arachidonate metabolites (12R-hydroxyeicosatetraenoic acid (12R-HETE), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), or 20-HETE), or lipoxygenase metabolites (5S-HETE, leukotriene B4, or lipoxin A4). We found that their metabolically more stable sulfonimide (SI) analogs (11,12-EET-SI and 14,15-EET-SI) were also potent mitogens. In addition 14,15-EET-SI also increased cell proliferation as well as expression of both c-fos and egr-1 mRNA. The protein kinase C and A inhibitors, H-7 and H-8, or the cyclooxygenase inhibitor, indomethacin, had no effect upon 14, 15-EET-induced [3H]thymidine incorporation, but the selective tyrosine kinase inhibitor, genistein, significantly inhibited it. Immunoprecipitation and immunoblotting demonstrated increased tyrosine phosphorylation of PI3-kinase and epidermal growth factor receptor (EGFR) within 1 min of EET administration. EETs also stimulated association of PI3-kinase with EGFR. PI3-kinase inhibitors, wortmannin and LY 294002, markedly inhibited 14, 15-EET-SI-stimulated [3H]thymidine incorporation. In addition, 14, 15-EET-SI administration stimulated tyrosine phosphorylation of src homologous and collagen-like protein (SHC) and association of SHC with both growth factor receptor-binding protein (GRB2) and EGFR. Mitogen-activated protein kinase was also activated within 5 min. Pretreatment of the cells with the mitogen-activated protein kinase kinase inhibitor, PD98059, inhibited the 14,15-EET-SI-stimulated [3H]thymidine incorporation. Moreover, immunoblotting indicated that 14,15-EET stimulated tyrosine phosphorylation of the specific pp60(c-src) substrate p120 and c-Src association with EGFR. 14, 15-EET increased src kinase activity within 1 min. Our data indicate that EETs are potent mitogens for renal epithelial cells, and the mitogenic effects of the EETs are mediated, at least in part, by the activation of Src kinase and initiation of a tyrosine kinase phosphorylation cascade.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

Distinct WNT pathways regulating AER formation and dorsoventral polarity in the chick limb bud.

The apical ectodermal ridge (AER) is an essential structure for vertebrate limb development. Wnt3a is expressed during the induction of the chick AER, and misexpression of Wnt3a induces ectopic expression of AER-specific genes in the limb ectoderm. The genes beta-catenin and Lef1 can mimic the effect of Wnt3a, and blocking the intrinsic Lef1 activity disrupts AER formation. Hence, Wnt3a functions in AER formation through the beta-catenin/LEF1 pathway. In contrast, neither beta-catenin nor Lef1 affects the Wnt7a-regulated dorsoventral polarity of the limb. Thus, two related Wnt genes elicit distinct responses in the same tissues by using different intracellular pathways.

Amino Acid Sequence↗
PubMed1998

Endogenous and ectopic expression of noggin suggests a conserved mechanism for regulation of BMP function during limb and somite patterning.

The gene noggin, originally cloned in Xenopus, encodes a secreted factor expressed in the Spemann organizer, where it functions to oppose the ventralizing influence of bone morphogenetic proteins (BMPs). Noggin protein acts by binding directly to BMPs, thereby preventing them from interacting with their receptors. Here we describe the pattern of expression of the chicken noggin gene during somite and limb development, two tissues in which BMPs have been postulated to play essential patterning roles. We find that noggin is expressed in dynamic restricted patterns consistent with an important role in the modulation of BMP signaling. Using a replication competent retrovirus we have ectopically expressed noggin in developing somitic and limb bud mesoderm and observed phenotypes consistent with complete block of BMP activity. This includes suppression of lateral somite differentiation and, in the limb, complete inhibition of chondrogenesis and local suppression of programmed cell death. In addition, we find that ectopic noggin expression in the limb has no effect on anteroposterior limb pattern, suggesting that BMPs are unlikely to play a significant role in this process. Taken together,, our results indicate that noggin is a key regulator of vertebrate limb and somite patterning and suggest that the antagonistic Noggin-BMP interaction is a widely used mechanism to modulate BMP signaling during multiple inductive events in vertebrate embryogenesis.

Animals↗
PubMed1998

Control of dorsoventral somite patterning by Wnt-1 and beta-catenin.

In vertebrates, the dorsoventral patterning of somitic mesoderm is controlled by factors expressed in adjacent tissues. The ventral neural tube and the notochord function to promote the formation of the sclerotome, a ventral somite derivative, while the dorsal neural tube and the surface ectoderm have been shown to direct somite cells to a dorsal dermomyotomal fate. A number of signaling molecules are expressed in these inducing tissues during times of active cell fate specification, including members of the Hedgehog, Wnt, and BMP families. However, with the exception of the ventral determinant Sonic hedgehog (Shh), the functions of these signaling molecules with respect to dorsoventral somite patterning have not been determined. Here we investigate the role of Wnt-1, a candidate dorsalizing factor, in the regulation of sclerotome and dermomyotome formation. When ectopically expressed in the presomitic mesoderm of chick embryos in ovo, Wnt-1 differentially affects the expression of dorsal and ventral markers. Specifically, ectopic Wnt-1 is able to completely repress ventral (sclerotomal) markers and to enhance and expand the expression of dorsal (dermomyotomal) markers. However, Wnt-1 appears to be unable to convert all somitic mesoderm to a dermomyotomal fate. Delivery of an activated form of beta-catenin to somitic mesoderm mimics the effects of Wnt-1, demonstrating that Wnt-1 likely acts directly on somitic mesoderm, and not through adjacent tissues via an indirect signal relay mechanism. Taken together, our results support a model for somite patterning where sclerotome formation is controlled by the antagonistic activities of Shh and Wnt signaling pathways.

Animals↗