Signal Transduction Mechanisms About The Effects Of Transforming Growth Factor Beta 2 On Human Lens Epithelial Cells Apoptosis And Invasiveness

Cataract is the leading cause of blindness worldwide. There are still no medicine or threptic methods which can cure or delay the progress of cataract.The treatment for cataract is routine, surgical removal of cataracts and implantation of replacement lenses so far. But there are still many questions after the operation, such as loss of accommodation; after cataract and the possibility of intraocular inflammation, which would degrade the therapeutic effect. It is important to point out that the case for further treatment are always young especial children, and those who have diabetic retinopathy or traumatic cataract. It is important to clarify the mechianism of cataract and after cataract for searching more safe and effective therapeutic methods.Transforming growth factor β (TGF-β) has recently been identified as a criticalregulator of many pathological growth conditions in the lens. For example, TGF-β has been shown to induce anterior sub-capsular cataract (ASC) in a rat lens culture model and analysis of human tissue has created a body of evidence that strongly implicate TGF-β in this process also. TGF-β is also now being examined as a causative factor in another growth condition of the lens. Posterior capsule opacification (PCO), which arises from vigorous lens cell growth following cataract surgery. Recent work has provided useful evidence that not only TGF-β and its receptors are present in capsular bags following surgery, but also some proteins which are strongly regulated by TGF-β, including connective tissue growth factor (CTGF).At least five isoforms of TGF-β have been reported with TGF β- 1, 2 and 3 being present in mammals. In the eye, altered intraocular fluid concentrations of TGF-β have been reported in the context of various ocular conditions and pathologies. The major isoform synthesised within the eye is TGF-β₂. In addition to TGF-β₂ two further isoforms of TGF-β have been identified, TGF-β₁ and TGF-β₃. Both isoforms play an only minor role in immunomodulation of the anterior ocular segment.Transforming growth factor-β₂ (TGF-β₂) is an approximately 25 kDa polypeptide encoded by a unique gene located on chromosome lq41. This dimeric peptide is ubiquitously distributed in human tissues and synthesized by many different human cells. It has been detected in tear fluid, in the vitreous, and in aqueous humor. TGF-β₂ is known to play an important role in wound healing and the production of the extracellular matrix. It inhibits cell proliferation and exerts various immunosuppressive effects. In the anterior chamber, TGF-β₂ is relevant for the maintenance of an immunosuppressive climate, as it alters the activities of antigen-presenting cells, and suppresses T-cell proliferation, IFN-γ production, and the inflammatory activity of macrophages. It is secreted as an inactive precursor (latent TGF-β₂ complex, LTGF-β₂) by cell types such as corneal endothelial cells, cells of the trabecular meshwork, and the ciliary body. This precursor (200 kDa) is complexed with latency-associated peptide (LAP) and bound to latent TGF-β binding protein (LTBP). L-TGF-β₂ is not able to bindto its receptor until LAP and LTBP are removed extracellularly via proteolytic cleavage. The exact mechanisms by which latent TGF-β₂ is activated physiologically are not completely understood. One model of activation has been proposed in which latent TGF-β is released from the extracellular matrix by proteases, localized to cell surfaces, and activated for example by thrombospondin-1 or specific integrins. Following this activation, TGF-β₂ exerts its biological functions via binding to a membrane-bound heteromeric receptorDifferent levels of total TGF-β₂ have been found in human aqueous humor, depending on the ocular disorders concerned. The ratio of active to total TGF-β₂ in the aqueous humor has been reported to be between 11 to 61% Tripathy et al. found active TGF-β₂ levels in cataract patients of 200±240 pg/ml (range 20-830 pg/ml), and Jampel et al. found a ratio of active-to-total TGF-β₂ of 61% for cataract patients.Previous work have revealed that TGF-β play an important role in ASC and PCO. The mechanisms about the effect of TGF-β on lens are studied extensively. In this study, we started with the investigation of aoptosis in human lens epithelail cells induced by TGF-β₂. We found that reactive oxygen species (ROS) mediated this process and a majority of cells kept an vigorous acvitity to invasiveness. A further research found that integrin β₁ took part in this process. This study performed a deeply investigation about the effect of TGF-β on human lens epithelail cells from two aspects. All these work will benefit for reveal the mechanism about the effect of TGF-β on lens in and physiological and pathological conditions.Reactive oxygen species (ROS) mediate the apoptosis induced by transforming growth factor beta 2 in human lens epithelial cells[Objective]To investigate the possible role of reactive oxygen species (ROS) in the apoptotic process induced by transforming growth factor beta 2 (TGF-β₂) in human lens epithelial cells (HLECs).[Methods]HLE B-3 cells were treated with different concentrations of TGF-β₂. MTT assay and TUNEL assay were used to analyze cell proliferation and apoptosis, respectively. We used DCFH-DA, an oxidation-sensitive fluorescent probe to examine the generation of ROS in HLECs that were treated with TGF-β₂. Furthermore, we investigated cells for glutathione and c-fos mRNA intracellular levels.[Results]Transforming growth factor-β₂, a growth regulator of HLECs in culture, also regulates the death of these cells. Dose-response analysis showed that the TGF-β₂ concentration needed to induce HLEC death (100 pg/ml) was 10 times that needed to inhibit growth in these cells (10 pg/ml). TGF-β₂-induced apoptosis in HLECs was preceded by an induction of ROS and a decrease in glutathione in the intracellular content, indicating that this factor induces oxidative stress in HLECs. Studies performed to analyze the levels of c-fos mRNA, a gene whose expression is modulated by the redox state, demonstrated that only high, apoptotic concentrations of TGF-β₂ (100 pg/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of hydrogen peroxide. Finally, the cell death induced by TGF-β₂ in HLECs was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells,whereas these agents did not modify the growth-inhibitory effect elicited by TGF-β₂ in these cells.[Conclusion]The results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-β₂ in HLECs.Part IIIntegrin β1 and Integrin-related signaling are necessary for transforming growth factor beta 2-promoted invasiveness inhuman lens epithelial cells[Objective]In this study, we investigate the possible implication of integrin and integrin-related signaling in TGF-β₂-promoted invasiveness in human lens epithelial cells.[Methods]A human lens epithelial cell line (HLE B-3) was treated with 100 pg/ml TGF-β₂ for 6, 12, 24h respectively. In vitro wound healing assay and cell adhesion assay were perfromed to detected the effect of TGF-β₂ on HELCs adhesion and migration. Integrin β₁ expression changes in HELCs during the treatment of TGF-β₂ were detected in protein level and mRNA level using confocal microscopy, flow cytometric analysisand real time quantitative reverse transcription polymerase chain reaction (RT-PCR) respectively. Focal adhesion kinase (FAK) activity was examined by FAK phosphorylation (Tyr 576) and total tyrosine phosphorylation during treatment with TGF-β₂ in HLEC. [Results]In this study, we found that TGF-β₂ significantly stimulated cell adhesion and migration in HLECs. By immunofluorescence staining and Western blotting, we observed that TGF-β₂ markedly enhanced the expression of integrin β₁ and the Tyr-Phosphorylation of focal adhesion kinase (FAK). Real time quantitative RT-PCR also showed the mRNA level of integrin β₁ upregulated. Neutralizing anti-integrin β₁ monoclonal antibody inhibited TGF-β₂-promoted HLECs adhesion and migration significantly(P<0.05).[ Conclusion ]TGF-β₂ promoted HLECs adhesion and migration in vitro. Integrin β₁ and integrin-related signaling are necessary for TGF-β₂-promoted adhesion and migration in human lens epithelial cells.SummaryThe multifunctional growth factor transforming growth factor β (TGF-β) is one of the most important ligands involved in modulation of cell behavior in ocular tissues. TGF-β, especially TGF-β₂, which is the predominant cytokine, involved in the regulation of cell behavior in lens in physiological or pathological processes of development or tissue repair, although various other growth factors are also involved. In this article, we found that TGF-β₂ induced supression of human lens eipthelail cells in time and concentration manner. Moreover, it induced cells underwent apoptosis which correlated with some types of cataract formation. Reactive oxygen species involved in and modulated this process.Furthermore, we found that a majority of cells survived. We found that TGF-β₂ stimulated integrin β₁ expression and the phosphorylation of focal adhesion kinase, which is the downstream of integrin signal. TGF-β₂ promoted human lens epitheial cells adhesion and migration through this signal. The results presented made a progress to clarify the mechinasm of TGF-β₂ induced posterior capsular opacification formation.