Analysis of the calcium-dependent regulation of proline-rich tyrosine kinase 2 by gonadotropin-releasing hormone.

Calcium influx through L-type voltage-gated calcium channels (VGCC) is required for ERK activation induced by GnRH in pituitary gonadotropes. The current studies investigate VGCC-sensitive catalytic activities that may lie upstream of ERKs within the GnRH signaling network. Ion exchange fractionation of alphaT3-1 cell lysates subjected to anti-phosphotyrosine Western blot analysis revealed a nifedipine-sensitive activity that colocalized with proline-rich tyrosine kinase (Pyk) 2 immunoreactivity. Phosphorylated Pyk2 was present in alphaT3-1 cells after GnRH agonist administration for a time course that lasted up to 4 h. Pyk2 phosphorylation was also evident in gonadotropes in vivo after administration of a bolus of GnRH. Knockdown of Pyk2 using specific small interfering RNAs revealed that Pyk2 contributed to modulation of GnRH-induced ERK but not c-Jun N-terminal kinase activation. Using pharmacological approaches, calmodulin (Cam) was also demonstrated to be required for the phosphorylation of Pyk2. Pyk2 was shown to bind specifically to a Cam agarose affinity column in a calcium-dependent manner, suggesting Cam and Pyk2 are capable of forming a complex. Specific mutation of a putative Cam binding motif within the catalytic domain of Pyk2 blocked association with Cam and uncoupled Pyk2's ability to activate ERK-dependent gene transcription. Thus, GnRH induces Pyk2 tyrosine phosphorylation dependent upon calcium flux within gonadotropes. Furthermore, association of Pyk2 and Cam may be required to mediate the effects of calcium on Pyk2 phosphorylation and subsequent activation of ERKs by GnRH.

Analysis of the gene regulatory program induced by the homeobox transcription factor distal-less 3 in mouse placenta.

Dlx3, a homeodomain transcription factor, is essential for placental development in the mouse. The Dlx3(-/-) mouse embryo dies at embryonic d 9.5-10 putatively due to placental failure. To develop a more comprehensive understanding of the gene profile regulated by Dlx3, microarray analysis was used to determine differences in gene expression within the placenta of Dlx3(+/+) and Dlx3(-/-) mice. Array analysis revealed differential expression of 401 genes, 33 genes in which signal to log ratio values of null/wild-type were lower than -0.5 or higher than 0.5. To corroborate these findings, quantitative real-time PCR was used to confirm differential expression for 11 genes, nine of which displayed reduced expression and two with enhanced expression in the Dlx3(-/-) mouse. Loss of Dlx3 resulted in a marked reduction (>60%) in mRNA expression of placental growth factor (Pgf), a member of the vascular endothelial growth factor family. Consistent with these results, Pgf secretion from placental explants tended to be reduced in the Dlx3(-/-) mice, compared with wild type. To investigate mechanisms of Dlx3 regulation of Pgf gene transcription, we cloned 5.2 kb of the Pgf 5' flanking sequence for use in reporter gene assays. Expression of the Pgf promoter luciferase reporter containing at least three Dlx3 binding sites was increased markedly by overexpression of Dlx3 supporting the conclusion that Dlx3 may have a direct effect on Pgf promoter activity. These studies provide a novel view of the transcriptome regulated by Dlx3 in mouse placenta. Dlx3 is specifically required for full expression and secretion of Pgf in vivo. Moreover, in vitro studies support the conclusion that Dlx3 is sufficient to directly modulate expression of the Pgf gene promoter in placental cells.

Smad6 represses Dlx3 transcriptional activity through inhibition of DNA binding.

Dlx3 (Distal-less 3) is a homeobox-containing transcription factor required for normal placental development in mice. Here we demonstrate that Dlx3 interacts with Smad6, a member of a larger family of transcriptional regulators generally thought to regulate transforming growth factor beta/bone morphogenetic protein signaling. Immunocytochemical and immunoprecipitation studies demonstrate overlapping nuclear localization and physical interaction between Dlx3 and Smad6 in human choriocarcinoma cells and in differentiated trophoblasts from human placenta. In vitro protein interaction studies mapped the Smad6 interaction domain within Dlx3 to residues 80-163, a region of Dlx3 that includes a portion of the homeodomain. Dlx3 and Dlx4 share homology within this region, and Dlx4 was also found to bind Smad6. Using the Esx1 gene promoter as a model for a Dlx3-responsive gene, studies demonstrate two near consensus Dlx3 binding sites within the proximal 2.3 kb of the transcription start site. Interestingly, binding of Dlx3 to one of these two sites was inhibited by interaction with Smad6. Consistent with this result, expression of an Esx1 promoter luciferase reporter was increased by overexpression of Dlx3; this effect was reversed with co-expression of Smad6. Further, small interference RNA-mediated knockdown of endogenous Smad6 increased Dlx3-dependent expression of the Esx1 gene promoter. Thus, Smad6 appears to functionally interact with Dlx3, altering the ability of Dlx3 to bind target gene promoters. Smad6 appears to play a modulatory role in the regulation of Dlx3-dependent gene transcription within placental trophoblasts.

Intercalation, delivery, and expression of the gene encoding green fluorescence protein utilizing nanobiohybrids.

Non-viral gene vectors have attracted much attention recently due to setbacks with viral delivery systems. Here a new non-viral delivery system based on nanobiohybrids synthesized by the intercalation of a full gene and promoter encoding Green Fluorescent Protein (GFP) between the layers of an inorganic host is reported. The nanobiohybrids were delivered to 9L glioma cells, JEG3 choriocarcinoma placental cells, and cardiac myocytes. All cells were able to internalize and tolerate the nanobiohybrids. In addition, all cells expressed the gene with some cell lines having up to 90% transfection efficiency. This new, bio-mimetic delivery system shows promise for use in non-viral gene therapy.

A role for CCAAT/enhancer-binding protein beta in the basal regulation of the distal-less 3 gene promoter in placental cells.

The homeodomain protein Distal-less 3 (Dlx3) is essential for normal placental development in mice. Dlx3-null mice die by embryonic day 10.0 due to placental failure. The aim of our studies was to examine the transcriptional regulation and expression of Dlx3 in choriocarcinoma cell lines and primary trophoblasts from human placenta. A Dlx3 promoter fragment coupled to a luciferase reporter gene was sufficient to increase luciferase activity more than 11-fold over a luciferase control vector in choriocarcinoma cells, but not in a heterologous gonadotrope cell line. A 5' deletion series of the Dlx3 promoter revealed that a 13-nucleotide CCAAT box-containing element was required for basal expression in choriocarcinoma cell lines. Mutation of the CCAAT box within the context of the full-length promoter resulted in reduced basal activation of the Dlx3 reporter gene, suggesting that the CCAAT box was required for full basal expression. Western blot analysis revealed that Dlx3, CCAAT/enhancer-binding protein alpha (C/EBP alpha), and C/EBP beta were present in choriocarcinoma cells and isolated trophoblasts from term human placentas. Electrophoretic mobility shift assays revealed the formation of a specific complex between choriocarcinoma cell nuclear extracts and the Dlx3 CCAAT box sequence. Competition and antibody electrophoretic mobility shift assays revealed that CCAAT/enhancer-binding protein beta (C/EBP beta) binds the Dlx3 CCAAT box sequence. Overexpression of C/EBP beta was sufficient to increase basal expression of a Dlx3 reporter gene in a dose-dependent manner. These studies provide the first insight into the mechanism(s) of Dlx3 gene expression in placental cells and suggest a role for C/EBP beta in the basal regulation of the Dlx3 gene.

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK.

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.