o Lu

Activation of Polo-like Kinase 3 by Hypoxic Stresses

Hypoxia/reoxygenation stress induces the activation of specific signaling proteins and activator protein 1 (AP-1) to regulate cell cycle regression and apoptosis. In the present study, we report that hypoxia/reoxygenation stress activates AP-1 by increasing c-Jun phosphorylation and DNA binding activity through activation of Polo-like-kinase 3 (Plk3) resulting in apoptosis. The specific effect of hypoxia/reoxygenation stress on Plk3 activation resulting in c-Jun phosphorylation was the opposite of UV irradiation-induced responses that are meanly independent on activation of the stress-induced JNK signaling pathway in human corneal epithelial (HCE) cells. The effect of hypoxia/reoxygenation stress-induced Plk3 activation on increased c-Jun phosphorylation and apoptosis was also mimicked by exposure of cells to CoCl2. Hypoxia/reoxygenation activated Plk3 in HCE cells to directly phosphorylate c-Jun proteins at phosphorylation sites Ser-63 and Ser-73, and to increase DNA binding activity of c-Jun, detected by EMSA. Further evidence demonstrated that Plk3 and phospho-c-Jun were immunocolocalized in the nuclear compartment of hypoxia/reoxygenation stress-induced cells. Increased Plk3 activity by overexpression of wild-type and dominantly positive Plk3 enhanced the effect of hypoxia/reoxygenation on c-Jun phosphorylation and cell death. In contrast, knocking-down Plk3 mRNA suppressed hypoxia-induced c-Jun phosphorylation. Our results provide a new mechanism indicating that hypoxia/reoxygenation induces Plk3 activation instead of the JNK effect to directly phosphorylate and activate c-Jun, subsequently contributing to apoptosis in HCE cells.

Function of GABA receptor/channel subunits in spinal cord

γ-Aminobutyric acid (GABA) receptor/channel ρ1 subunits are important components in inhibitory pathways in the central nervous system. However, the precise locations and roles of these receptors in the central nervous system are unknown. We studied the expression localization of GABA receptor/channel ρ1 subunit in mouse spinal cord and dorsal root ganglia (DRG). The immunohistochemistry results indicated that GABA receptor/channel ρ1 subunits were expressed in mouse spinal cord superficial dorsal horn (lamina I and lamina II) and in DRG. To understand the functions of the GABA receptor/channel ρ1 subunit in these crucial sites of sensory transmission in vivo, we generated GABA receptor/channel ρ1 subunit mutant mice (rho1-/-). GABA receptor/channel ρ1 subunit expression in the rho1-/- mice was eliminated completely, whereas the gross neuroanatomical structures of the rho1-/- mice spinal cord and DRG were unchanged. Electrophysiological recording showed that GABA-mediated spinal cord response was altered in the rho1-/- mice. A decreased threshold for mechanical pain in the rho1-/- mice compared with control mice was observed with the von Frey filament test. These findings indicate that the GABA receptor/channel ρ1 subunit plays an important role in modulating spinal cord pain transmission functions in vivo.

Hyperosmotic Stress-induced ATF-2 Activation through Polo-like Kinase 3 in Human Corneal Epithelial Cells

Elevated extracellular solute concentration (hyperosmotic stress) perturbs cell function and stimulates cell responses by evoking MAPK cascades and activating AP-1 transcription complex resulting in alterations of gene expression, cell cycle arrest, and apoptosis. The results presented here demonstrate that hyperosmotic stress elicited increases in ATF-2 phosphorylation through a novel Polo-like kinase 3 (Plk3) pathway in human corneal epithelial (HCE) cells. We found in hyperosmotic stress-induced HCE cells that Plk3 transferred to the nuclear compartment and was colocalized with ATF-2 in nuclei. Kinase activity of Plk3 was significantly activated by hyperosmotic stimulation. Further downstream, active Plk3 phosphorylated ATF-2 at the Thr-71 site in vivo and in vitro. Overexpression of Plk3 and its mutants enhanced hyperosmotic stress-induced ATF-2 phosphorylation. In contrast, suppression of Plk3 by knocking down Plk3 mRNA effectively diminished the effect of hyperosmotic stress-induced ATF-2 phosphorylation. The effect of hyperosmotic stress-induced activation of Plk3 on ATF-2 transcription factor function was also examined in CRE reporter-overexpressed HCE cells. Our results for the first time reveal that hyperosmotic stress can activate the Plk3 signaling pathway that subsequently regulates the AP-1 complex by directly phosphorylating ATF-2 independent from the effects of JNK and p38 activation.

Hyperosmotic stress-induced corneal epithelial cell death through activation of Polo-like kinase 3 and c-Jun.

PURPOSE Hyperosmotic stress causes cell shrinkage, perturbs cell function, and damages DNA, resulting in cell cycle arrest and apoptosis. In the present study, the authors explore the mechanism involving hyperosmotic stress-induced activation of c-Jun/AP-1 through a novel Plk3 pathway in human corneal epithelial cells. METHODS Human primary corneal epithelial cells and cell line were cultured in a serum-free keratinocyte medium and DMEM/F12 medium containing 10% FBS in a 37°C incubator supplied with 5% CO(2), respectively. Western blot analysis was used to determine protein expression and phosphorylation levels. Protein kinase activities were measured by immunocomplex kinase assay. Cell viability and apoptosis were determined by MTT assay and caspase-3 (DEVDase) activity. RESULTS It was found that hyperosmotic stress-induced increases in the phosphorylation of c-Jun, resulting in apoptosis through the activation of Plk3 in human corneal epithelial cells. Plk3 was activated by extracellular hyperosmotic stress to directly phosphorylate c-Jun in the serine 63 and 73 residues. Hyperosmotic stress-induced c-Jun phosphorylation was enhanced by overexpression of constitutively positive Plk3 mutants and suppressed by the knockdown of Plk3 mRNA with Plk3-specific siRNA. Further studies indicated that the phosphorylation of c-Jun by Plk3 was responsible for hyperosmotic stress-induced apoptosis, which was independent from activation of the JNK signaling pathway in human corneal epithelial cells. CONCLUSIONS These results, for the first time, provide a novel and alternative signaling mechanism that involves hyperosmotic stress-induced activation of the Plk3 pathway in addition to JNK/p38 MAPK pathways to regulate the c-Jun/AP-1 transcriptional complex and human corneal epithelial cell fate.

Stress-induced c-Jun Activation Mediated by Polo-like Kinase 3 in Corneal Epithelial Cells

Polo-like kinase 3 (Plk3) activation occurs after exposure to environmental or genotoxic stresses. Plk3 regulates cell fate through regulating cell cycle progression. UV irradiation is one of the major environmental stresses that affect corneal epithelial wound healing. In the present study, we report that UV irradiation activated Plk3 and that Plk3 interacts with AP-1 and c-Jun, which appears to be important to mediate corneal epithelial cell apoptosis after UV irradiation. Recombinant Plk3, as well as Plk3 immunoprecipitated from UV-irradiated cells, phosphorylated c-Jun in vitro. The phosphorylation of c-Jun by Plk3 immunoprecipitates was not altered by the pre-removal of JNK from the cell lysates. In addition, the effect of UV irradiation-induced phosphorylation of c-Jun and apoptosis were not significantly affected by knockdown of JNK mRNA. Co-immunoprecipitation reveals that Plk3 and c-Jun directly interacted with each other. Consistently, Plk3 co-localized with c-Jun to the nucleus after UV irradiation. Further, modulating Plk3 activities by overexpressing Plk3 or its mutants significantly affected UV irradiation-induced c-Jun activity and subsequent apoptosis. Our results thus provide for the first time that Plk3 mediates UV irradiation-induced c-Jun activation by phosphorylating c-Jun, suggesting that Plk3 plays an important role in mediating programmed cell death of corneal epithelial cells after UV irradiation.

De-SUMOylation of CCCTC Binding Factor (CTCF) in Hypoxic Stress-induced Human Corneal Epithelial Cells

Background: CCCTC binding factor (CTCF) plays important roles in the epigenetic control of cell fate. Results: Hypoxic stress suppressed a higher Mr form of CTCF by de-SUMOylation associated with lysine 74 and 689 residues, resulting in significantly inhibited PAX6 expression. Conclusion: Hypoxic stress induces de-SUMOylation of CTCF to functionally regulate CTCF activity. Significance: CTCF plays important roles in growth factor/stress-regulated cell fates through post-translational modulation to control gene expression. Epigenetic factor CCCTC binding factor (CTCF) plays important roles in the genetic control of cell fate. Previous studies found that CTCF is positively and negatively regulated at the transcriptional level by epidermal growth factor (EGF) and ultraviolet (UV) stimulation, respectively. However, it is unknown whether other stresses modify the CTCF protein. Here, we report that regulation of CTCF by de-SUMOylation is dependent upon hypoxic and oxidative stresses. We found that stimulation of human corneal epithelial cells with hypoxic stress suppressed a high molecular mass form of CTCF (150 kDa), but not a lower molecular weight form of CTCF (130 kDa). Further investigation revealed that the hypoxic stress-suppressed 150-kDa CTCF was a small ubiquitin-related modifier (SUMO)ylated form of the protein. Hypoxic stress-induced de-SUMOylation of human CTCF was associated with lysine 74 and 689 residues, but not to the phosphorylation of CTCF. Overexpression of SENP1 induced de-SUMOylation of CTCF. However, knockdown of SENP1 could not rescue hypoxic stress-induced CTCF de-SUMOylation. Overexpression of SUMO1 and SUMO2 increased SUMOylation of CTCF and partially blocked hypoxic stress-induced CTCF de-SUMOylation, suggesting that free cellular SUMO proteins play roles in regulating hypoxia-induced CTCF de-SUMOylation. In addition, hypoxic stress significantly inhibited PAX6 mRNA and protein expressions by suppression of PAX6-P0 promoter activity. The result was further supported by data showing that knockdown of CTCF significantly enhanced expression of PAX6 and abolished hypoxia-induced suppression of PAX6. Thus, we conclude that hypoxic stress induces de-SUMOylation of CTCF to functionally regulate CTCF activity.

Regulation of Pax6 by CTCF during Induction of Mouse ES Cell Differentiation

Pax6 plays an important role in embryonic cell (ES) differentiation during embryonic development. Expression of Pax6 undergoes from a low level to high levels following ES cell differentiation to neural stem cells, and then fades away in most of the differentiated cell types. There is a limited knowledge concerning how Pax6 is regulated in ES cell differentiation. We report that Pax6 expression in mouse ES cells was controlled by CCCTC binding factor (CTCF) through a promoter repression mechanism. Pax6 expression was significantly enhanced while CTCF activity was kept in the constant during ES cell differentiation to radial glial cells. Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation. Reduced occupancy of CTCF in the binding motif region upstream from the P0 promoter was due to increased DNA methylations in the CpG sites identified in the region. Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression. We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively.

Effect of EGF-Induced HDAC6 Activation on Corneal Epithelial Wound Healing

PURPOSE Epidermal growth factor (EGF) stimulates migration in corneal epithelial wound healing. The purpose of this study was to investigate the effect of EGF-induced alpha-tubulin deacetylation through activating HDAC6 on migration in corneal epithelial wound healing. METHODS Human corneal epithelial (HCE) cells were cultured in DMEM/F12 medium containing 10% FBS in a 37 degrees C incubator supplied with 5% CO(2). Western blot analysis was used to determine protein expression. Activity of HDAC6 was suppressed by trichostatin A (TSA) and by siRNA specific to HDAC6. Corneal epithelial cell migration was measured by using scratch-induced directional migration assay in cultured cells and by corneal epithelial debridement using a mouse whole-eye organ culture model. RESULTS The authors found EGF stimulated corneal epithelial cell migration in wound healing by enhancing HDAC6 activity, resulting in the deacetylation of alpha-tubulin. EGF stimulated HDAC6 enzymatic activity and protein translocation toward the leading edge of the cell. Inhibition of HDAC6 activity by TSA significantly suppressed EGF-induced cell migration and delayed EGF-induced wound healing in epithelially debrided mouse corneas. In the meantime, knockdown of HDAC6 mRNA with specific siRNA effectively abolished EGF-induced deacetylation of alpha-tubulin, resulting in the inhibition of cell migration. CONCLUSIONS These results reveal an important mechanism that involves EGF-induced HDAC6 activation and alpha-tubulin deacetylation, subsequently affecting corneal epithelial migration in the wound-healing process.

Study on olfactory function in GABAC receptor/channel ρ1 subunit knockout mice

The GABAC receptor/channel rho1 subunit plays an important role in the inhibitory pathway and sensory processing in the retina and spinal cord. Although it was suggested that the rho1 subunit plays a role in olfactory sensations, the precise role of the rho1 subunit in olfactory sensory function is still not clear. In the present study, we report that olfactory function was significantly altered in rho1 subunit knockout (rho1-/-) mice compared to its wildtype counterpart. The rho1 subunit mRNA, detected by reverse transcription (RT)-PCR experiments, was expressed in the olfactory bulb of wild-type mice. Expression of rho1 subunit proteins in the olfactory bulb was detected by immunohistochemistry in mitral cells in the mitral cell layer. Neither mRNA nor proteins of the rho1 subunit were found in olfactory bulb neurons in rho1-/- mice. Alterations of olfactory function in rho1-/- mutant mice compared to their wildtype littermates were examined by olfactory behavioral test. We found that sensitivity to the smell of citral odorant in rho1-/- mice was significantly greater compared to that of wildtype mice. Our results indicate that the GABAC rho1 subunit acts in olfactory bulb neurons as an inhibitory modulator that affects the process of olfactory signaling transmission.

Effect of Hypoxic Stress–Activated Polo-like Kinase 3 on Corneal Epithelial Wound Healing

PURPOSE Hypoxia/reoxygenation conditions can generate oxidative stresses resulting in the suppression of cell proliferation and the delay of corneal epithelial wound healing. The purpose of this study was to investigate the cellular mechanism involving the role of the stress-responsive Polo-like kinase 3 (Plk3) in hypoxic stress-induced delay of corneal epithelial wound healing. METHODS Plk3 activities were determined by immunochemistry and immunocomplex kinase assay approaches. Corneal epithelial wound healing was evaluated by a whole-eye organ culture model and by scratch-induced wound closure assay. Corneal epithelial layer was removed by using a corneal rust-ring-remover in wild-type and Plk3(-/-) mice. Wound healing was analyzed using a confocal imaging system. Cell growth was measured by MTT assays. RESULTS The effect of hypoxic stress on early stages of corneal epithelial wound healing was compared with other oxidative stresses, including UV, CoCl(2), and H(2)O(2) treatments. Hypoxic stress-induced delay of corneal epithelial wound healing was further evaluated in human corneal epithelial cells and in the corneas of wild-type and Plk3 knockout (Plk3(-/-)) mice. Hypoxic stress-induced Plk3 activation resulted in growth attenuation and delay of wound healing. Further evidence demonstrated that the increase in Plk3 activity in constitutively active Plk3-expressed cells significantly enhanced stress-induced delay of wound healing. In contrast, hypoxic stress-induced delay of wound healing was markedly diminished in the corneas of Plk3 deficient Plk3(-/-) mice. CONCLUSIONS These results provide for the first time important evidence that Plk3 plays a significant role in hypoxic stress-induced attenuation of cell growth and delay of corneal epithelial wound healing.