Abstract
Abstract: :
Purpose: Numerous studies have explored the role of TGFß2 in the initiation and progression of anterior uveitis. Much of this research has focused on TGFß2 levels in the aqueous environment and its participation in ocular immune privilege; however little is known about the regulation of TGFß2 via the Smad family of transcription factors during the disease. The purpose of our study was to evaluate and characterize the expression of TGFß2, receptor-mediated Smad, (Smad 2/3), common mediator Smad,( Smad 4), and the inhibitory element of TGFß2 expression, (Smad 7) during ocular inflammation. Methods: Endotoxin-induced uveitis (EIU) was induced in female Lewis rats by a footpad injection of LPS (250 ug). Control rats received saline. Animals were euthanized at time 0, 1, 3, 6, and 24 hours post injections. Eyes from control and EIU rats were enucleated and either formalin-fixed for immunohistochemistry (IHC) or the iris ciliary body (ICB) dissected. Dissected ICBs were placed in either Tri-Reagent for total RNA extraction or snap frozen on dry ice for protein extraction. Results: RT-PCR, Western analysis and densitometric assessment demonstrated invariant expression among time points for both TGFß2 and Smad 7. In contrast, temporal expression of both Smad2/3 and Smad 4 was seen. Immunolocalization studies via IHC revealed staining for Smad2/3, Smad 4, and Smad 7 in the ciliary epithelium. Conclusion: This study suggests, through expression characterization of TGFß2 and Smad 7, that despite the impact that inflammation has on the ICB, the regulation of TGFß2 within the ciliary epithelium-a predominant source of the cytokine, is not perturbed. These results further delineate the role of TGFß2 in the ICB and aqueous humor during uveitis. Furthermore, alterations in expression of Smad 2/3 and Smad 4 demonstrate the crosstalk which occurs with other signaling molecules (i.e., RSKs, RTKs) upregulated during the response. Further understanding the signaling mechanism will aid in elucidating the role of TGFß2 in anterior uveitis. Support: NIH grant #EY11364. CR: None
Keywords: 380 cytokines/chemokines • 605 transcription factors • 612 uveitis-clinical/animal model