Regulation Of Functions Of Lens Epithelial Cells By The Transcription Factor CREB And Its Phosphorylation Status At S133 Residue

Lens is the transmissive optical device in the eye which focuses the light reflected from cornea to retina and generate the observed image.The mature lens mainly comprises of two types of cells:the lens epithelial cells and the lens fiber cells,the later of which include both differentiating and organelle less terminally differentiated lens fiber cells.During the process of the lens fiber maturation,the organelles in the fiber cells will be degenerated.Thus,only the lens epithelial cells can divide and differentiate.The lens epithelial cells in the sub-equatorial region continuously divide and those moving to equatorial region will differentiate into lens fiber cells after exiting the cell cycle.While the stem cell-like lens epithelial cells in the center of the anterior lens constantly divide to supply the lens epithelial cells in the sub-equatorial region.Therefore,the lens epithelial cells play a key role in the lens development and growth.On the other hand,the metabolism of lens epithelial cells are most active in the whole lens,and play vital role in protecting the lens homeostasis and transparency.Furthermore,studies showing that the dysfunction or abnormal apoptosis of lens epithelial cells may trigger cataract and other ocular diseases.Therefore,study on lens epithelial cells is one of the most important subject of ocular development and pathogenesis.During lens development and normal physiological metabolism,a series of signals including fibroblast growth factor?FGF??autocrine bone morphogenetic protein?BMP?and notch signaling will induce different factors to be expressed in the specific region at the defined time,thus promoting lens development and maintaining normal functions.On the other hand,extensive studies revealed that the transcription factor Pax6plays a critical role in the regulation of lens development.Besides,the activity of Pax6 is regulated by the posttranslationalmodificationsincludingboth phosphorylation and SUMOylation.Furthermore,another well-known transcription factor CREB has also been identified as an important factor implicating in the control of lens development.Studies have shown that the phosphorylation of CREB at S133 is critical for its activation,and various protein kinases including protein kianse A?PKA?and mitogen-activated protein kinase?MAPK?can phosphorylate CREB at S133 to activate it.On the other hand,the studies in our lab revealed that the protein phosphatase 1and protein phosphatase 2A-C mediated CREB dephosphorylation,which plays an important role in the regulation of lens epithelial cells differentiation.However,the exact roles of CREB in regulating lens differentiation and the modulation of CREB functions by phosphorylation and dephosphorylation remain largely unknown.Thus,the major focus of this dissertation is to study the roles of CREB and the control of CREB functions by phosphorylation and dephosphorylation at S133 residue.In this thesis study,I first amplified the pCI-Neo vector?wild type CREB expression vector pCI-WT-CREB?constitutive dephosphorylation mutant expression vector pCI-S133A-CREB as well as the constitutive phosphorylation mutant expression vector pCI-S133D-CREB,and then purified these plasmids.The purified plasmids were transfected into the mouse lens epithelial cells?TN4-1.Through screening under G418 medium culture,four stable cell lines,pCI-Neo-?TN4-1?pCI-WT-CREB-?TN4-1?pCI-S133A-CREB-?TN4-1?pCI-S133D-CREB-?TN4-1 were established.There established stable cell lines together with the parent?TN4-1 cells were subsequently used in the later experimental studies.Using phase contrast microscope,the four stable cell lines as well as the parent?TN4-1 cells were examined morphologically.It was found that expression of exogenous genes altered their morphology.First,overexpression of wild type CREB caused elongation of the lens epithelial cells,thus became lens fiber-like cells.Futhermore,western blot results showed that the CREB phosphorylation at S133 increased its stability.In contrast,dephosphorylation at this site decreased its stability.Second,in order to study the roles of CREB and its phosphorylation at S133 in lens development,the effects of overexpression of wild type and mutant CREB on both proliferation and differentiation of lens epithelial cells were analyzed.Interestingly,my results revealed that expression of wild type CREB attenuates cell proliferation but promotes differentiation of lens epithelial cells.In contrast,neither the S133A mutant nor the S133D mutant transfected cell lines displayed the same effects.Furthermore,cultured in the serum free condition for 5 days,?TN4-1 cells expressing wild type CREB were induced to undergo differentiation and form the lentoid structure similar to those through bFGF-induction of the parent?TN4-1 cells,accompanied with the formation of lentoids,the differentiation marker,?-crystallin expression were observed.These data suggest that the role of the overexpressed wild type CREB is similar to the bFGF,both facilitating the differentiation of lens epithelial cells by promoting the cell cycle exit.Third,to explore the role of CREB and its phosphorylation at S133 in the stress response,okadaic acid?OA?was used to treat the cells and the relative apoptosis was analyzed by hoechst staining.The results revealed that overexpression of wild type CREB greatly enhanced apoptosis.This enhanced apoptosis is derived from down-regulated expressing of?B-crystallin by wild type CREB.Accordingly,overexpression of?B-crystallin in pCI-WT-CREB-?TN4-1 cells can rescue its survive in the okadaic acid condition.To demonstrate that CREB can directly regulate?B-crystallin expression.First,I searched the website and analyzed the possible CREB binding sites in the promoter of mouse?B gene.Potential CREB binding sites?CRE elements?were identified,and then tested by electrophoretic mobility shift assay.The results revealed that CREB can directly binds to?B-crystallin gene promoter through the sequence^-61TGACCTCA^-54.Next,the luciferase reporter gene assay was conducted to confirm that the wild type CREB binds to?B gene and negatively regulates its expression.In summary,the experimental data from this thesis research help to draw following major conclusions:first,I confirmed that phosphorylation of CREB promotes its stability,and the dephosphorylation decreased its stablilty.Second,for the first time,I demonstrated that wild type CREB has ability to promote the lens epithelial cell differentiation after driving them out of cell cycle.Third,for the first time,I discovered that wild type CREB inhibits the expression of?B-crystallin in lens epithelial cells,and thus leads to enhanced apoptosis of?TN4-1 cells expressing wild type CREB under stress condition?OA treatment here?.Fourth,my work demonstrate CREB can directly bind to the sequence^-61TGACCTCA^-54of the mouse?B gene promoter to suppress its expression.Last but not the least,this dissertation revealed that CREB phosphorylation status determines its functional partners,thus,wild type CREB?constitutive phosphorylation mutant and constitutive dephosphorylation mutant have strong phenotypes respectively which suggests the existence functional scope beyond pure phosphorylation.Together,my thesis provides novel information regarding the functional mechanisms of CREB and its control by phosphorylation and dephosphorylation associating with lens development.These information are also useful in understanding the mechanisms of lens pathogenesis.