Elena Zhukova

Protein kinase D2 potentiates MEK/ERK/RSK signaling, c-Fos accumulation and DNA synthesis induced by bombesin in Swiss 3T3 cells.

Protein kinase D (PKD) plays an important role in mediating cellular DNA synthesis in response to G protein‐coupled receptor (GPCR) agonists but the function of other isoforms of the PKD family has been much less explored. Here, we examined whether PKD2 overexpression in Swiss 3T3 cells facilitates DNA synthesis and the activation of the extracellular regulated protein kinase (ERK) pathway in response to the mitogenic GPCR agonist bombesin. We show that PKD2 overexpression markedly potentiated the ability of this agonist to induce DNA synthesis. Addition of bombesin to Swiss 3T3 cells overexpressing PKD2 also induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of control cells. In contrast, neither DNA synthesis nor the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD2‐independent pathways, was increased. Furthermore, bombesin promoted a striking accumulation of c‐Fos protein in cells overexpressing PKD2. Our study demonstrates that PKD2, like PKD, facilitates mitogenesis and supports the hypothesis that an increase in the duration of the ERK signaling leading to accumulation of immediate gene products is one of the mechanisms by which isoforms of the PKD family enhance re‐initiation of DNA synthesis by Gq‐coupled receptor activation. J. Cell. Physiol. 211: 781–790, 2007.

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells†

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small‐interfering RNA (siRNA)‐mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound‐induced migration and cytoskeletal organization in the non‐transformed intestinal epithelial cells IEC‐6 and IEC‐18 stimulated with the G protein‐coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline‐rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA‐induced migration of both IEC‐18 and IEC‐6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA‐transfected IEC‐18 cells. Interestingly, in contrast to the FAK−/− cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC‐18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA‐induced migration or cytoskeletal reorganization in IEC‐18 cells. In conclusion, our study shows that FAK plays a critical role in LPA‐induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA. J. Cell. Physiol.

CCKB/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin‐mediated mitogenesis, we have used high efficiency retroviral‐mediated transfection of cholecystokinin (CCK)B/gastrin receptor into Swiss 3T3 cells. The retrovirally‐transfected CCKB/gastrin receptor binds 125I‐CCK‐8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C‐dependent protein kinase D activation, and MEK‐dependent ERK1/2 activation. In the presence of insulin, CCK‐8 or gastrin induced a 66.5 ± 8.8‐fold (mean ± SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK‐8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCKB/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCKB/gastrin receptor retrovirally‐transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCKB/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.

Vasopressin-induced intracellular redistribution of protein kinase D in intestinal epithelial cells.

The spatio‐temporal changes of signaling molecules in response to G protein‐coupled receptors (GPCR) stimulation is a poorly understood process in intestinal epithelial cells. Here we investigate the dynamic mechanisms associated with GPCR signaling in living rat intestinal epithelial cells by characterizing the intracellular translocation of protein kinase D (PKD), a serine/threonine protein kinase involved in mitogenic signaling in intestinal epithelial cells. Analysis of the intracellular steady‐state distribution of green fluorescent protein (GFP)‐tagged PKD indicated that in non‐stimulated IEC‐18 cells, GFP‐PKD is predominantly cytoplasmic. However, cell stimulation with the GPCR agonist vasopressin induces a rapid translocation of GFP‐PKD from the cytosol to the plasma membrane that is accompanied by its activation via protein kinase C (PKC)‐mediated process and posterior plasma membrane dissociation. Subsequently, active PKD is imported into the nuclei where it transiently accumulates before being exported into the cytosol by a mechanism that requires a competent Crm1 nuclear export pathway. These findings provide evidence for a mechanism by which PKC coordinates in intestinal epithelial cells the translocation and activation of PKD in response to vasopressin‐induced GPCR activation. J. Cell. Physiol. 196: 483–492, 2003.

Gastrin and EGF synergistically induce cyclooxygenase-2 expression in Swiss 3T3 fibroblasts that express the CCK2 receptor.

Over‐expression of cyclooxygenase‐2 (COX‐2) has been demonstrated to be tumorigenic in transgenic mice. Chronic treatment with NSAIDs is chemoprotective for colorectal cancer. Gastrin is a growth factor for gastric mucosa and has been shown to promote proliferation of colorectal cells. Recent studies suggest that COX‐2 expression levels could mediate the growth effects of gastrin. Here, we report that gastrin increased PGE2 secretion in Swiss 3T3 cells expressing the CCK2 receptor. Gastrin dose dependently induced COX‐2 protein levels in a time dependent manner. COX‐2 mRNA levels were rapidly induced by a dose dependent increase in gastrin. Prior treatment of the cells with the CCK2 receptor specific antagonist, L365,260, inhibited gastrin‐induced COX‐2 protein and mRNA expression. Pretreatment with L364,714, the CCK1 receptor specific antagonist did not block COX‐2 induction by gastrin. Inhibition of de novo protein synthesis by cycloheximide did not block COX‐2 mRNA induction by gastrin. Also, gastrin‐dependent COX‐2 expression did not require PKC activity, activation of ERK, or transactivation of EGFR. However, co‐stimulation with EGF and gastrin synergistically induced COX‐2 protein and mRNA expression and PGE2 secretion. Measurements of COX‐2 mRNA stability and COX‐2 gene transcription reveal that EGF significantly increased the half‐life of COX‐2 mRNA with only a slight increase in the COX‐2 transcription rate. Conversely, gastrin significantly increased COX‐2 gene transcription rates but did not enhance COX‐2 mRNA stability. J. Cell. Physiol. 196: 454–463, 2003.

Expression of the human insulin gene in the gastric G cells of transgenic mice

The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.