Molecular Vision 2004; 10:703-11 <http://www.molvis.org/molvis/v10/a85> Received 19 August 2004 | Accepted 30 September 2004 | Published 4 October 2004 Transforming growth factor-β (TGF-β), a family of struc- turally related multifunctional cytokines, has wide biological actions including cell growth, differentiation, apoptosis, and fibrogenesis [1-3]. TGF-β typically is secreted in a latent form and is activated through a complex process of proteolytic ac- tivation and dissociation of latency protein subunits [4]. The action of TGF-β is mediated by TGF-β receptors types I (TβRI) and II (TβRII), both of which are serine and threonine kinases. Signal transduction is initiated by TGF-β binding to TβRII, followed by its association with TβRI [4]. TβRII phosphory- lates multiple serines and threonines in the TTSGSGSGSG sequence of the cytoplasmic region of TβRI [4]. The activated TβRI in turn phosphorylates and activates the transcription factors, Smads [4,5]. TGF-β has emerged as a key mediator of the fibrotic re- sponse to wounding. It is upregulated during different types of wound healing in the eye, liver, and skin [3,6-8]. Of the three human isoforms (TGF-β1, TGF-β2, and TGF-β3), TGF- β2 is the predominant form in the eye [9,10]. TGF-βs have been shown to be important in ocular scarring in conditions including proliferative vitreoretinopathy [11], cataract forma- tion [12], corneal opacities [13], and subconjunctival scarring, a complication of filtration surgery in glaucoma [14,15]. TGF- β also has been implicated in choroidal neovasculaization [16,17]. In glaucoma filtration surgery, postoperative scarring at the wound site is a critical determinant of the surgical out- come [18,19]. Although anti-scarring agents such as mitomy- cin C and 5-fluorouracil can prevent post-operative scarring and improve surgical outcome [20,21], they do so by causing widespread fibroblast cell death and are associated with se- vere and potentially blinding complications [22,23]. The im- portant role of TGF-β in the wound repair has led to other strategies [15] such as the use of anti-TGF-β antibody [24,25] and antisense oligonucleotides [26] to block the TGF-β ac- tion. These studies have, in general, targeted the ligand rather than the receptor. In the current study, we used the RNA interference (RNAi) strategy to target the TGF-β pathway. Gene silencing is a pro- cess by which double stranded RNA triggers the destruction of mRNAs sharing the same sequence. RNAi is initiated by the conversion of double stranded RNA into 21-23 nucleotide ©2004 Molecular Vision RNA interference targeting transforming growth factor-β type II receptor suppresses ocular inflammation and fibrosis Hiroshi Nakamura, 1 Shahid S. Siddiqui, 2 Xiang Shen, 1 Asrar B. Malik, 2 Jose S. Pulido, 1 Nalin M. Kumar, 1 Beatrice Y. J. T. Yue 1 (The first two authors contributed equally to this publication) Departments of 1 Ophthalmology and Visual Sciences, and 2 Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, IL Purpose: Transforming growth factor-β (TGF-β) is an important mediator of wound healing responses. In the eye, TGF- β activity has been implicated in causing corneal haze after laser surgery and subconjunctival scarring following glau- coma surgery. The purpose of the study was to determine whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-β (TβRII) could be used to suppress the TGF-β action. Methods: TβRII specific siRNAs designed from the human gene sequence were transfected into cultured human corneal fibroblasts. The protein and transcript levels of the receptor were determined by immunofluorescence, western blotting, and real time PCR. Immunofluorescence and immunoblotting were carried out to examine fibronectin assembly. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. In addition, the in vivo effects of TβRII siRNA were evaluated in a mouse model of ocular inflammation and fibrosis generated by subconjunctival injection of phosphate buffered saline and latex beads. Mouse TβRII siRNA was introduced into experimental eyes. Cellularity on tissue sections was evaluated after staining with hematoxylin and eosin. Collagen deposition was visualized by picrosirius red staining. Results: TβRII siRNAs abrogated the receptor transcript and protein expression in cultured corneal fibroblasts. The gene knockdown inhibited fibronectin assembly and retarded cell migration. In the mouse model, introduction of TβRII spe- cific siRNA significantly reduced the inflammatory response and matrix deposition. Conclusions: TβRII specific siRNAs were efficacious both in vitro and in vivo in knocking down the TGF-β action. A direct application of siRNA into eyes to downregulate TβRII expression may provide a novel therapy for preventing ocular inflammation and scarring. Correspondence to: Dr. Beatrice Yue, Department of Ophthalmol- ogy and Visual Sciences, University of Illinois at Chicago, 1855 West Taylor Street, Chicago, IL, 60612; Phone: (312) 996-6125; FAX: (312) 996-7773; email: Beatyue@uic.edu 703