TGFB1-induced extracellular expression of TGFBIp and inhibition of TGFBIp expression by RNA interference in a human corneal epithelial cell line.
OBJECTIVE
To report the increased production of extracellular transforming growth factor β-induced protein (TGFBIp) by human corneal epithelial cells (HCECs) after induction by TGFB1 and the inhibition of TGFBIp production in induced and noninduced HCECs by RNA interference (RNAi).


METHODS
HCECs were cultured in serum-free medium and treated with 0 or 10 ng/mL TGFB1 over a period of 72 hours. Commercially available siRNAs targeting TGFBI mRNA were mixed with a transfection reagent and used to reverse transfect TGFB1-induced and noninduced HCECs. Extracellular and intracellular concentrations of TGFBIp were measured by ELISA and Western blot analysis, respectively, and TGFBI RNA was assayed using semiquantitative RT-PCR.


RESULTS
HCECs constitutively express TGFBIp, and treatment with TGFB1 results in up to a fourfold increase in the amount of extracellular TGFBIp. Four commercially available siRNAs targeting TGFBI mRNA produced a >70% decrease in extracellular TGFBIp within 48 hours after transfection of noninduced HCECs but a <25% decrease in extracellular TGFBIp by 48 hours after transfection of TGFB1-induced HCECs. The suppression of extracellular TGFBIp production correlated with a decrease in intracellular TGFBIp production and TGFBI mRNA expression after transfection.


CONCLUSIONS
Extracellular TGFBIp expression by HCECs is increased several fold after exposure to TGFB1. Both HCEC-constitutive and HCEC-induced TGFBIp production can be inhibited with RNA interference, though the effect was greater and lasted longer for constitutive than induced TGFBIp production. Given that the corneal deposits in the TGFBI dystrophies consist of TGFBIp derived from HCECs, RNAi represents a potential means to inhibit primary dystrophic deposit formation and recurrence after surgical intervention.
Yellore
Vivek S
VS
Jules Stein Eye Institute, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA.
Rayner
Sylvia A
SA
Aldave
Anthony J
AJ
D015153:Blotting, Western; D002460:Cell Line; D002474:Cell-Free System; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D034622:RNA Interference / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D000099222:betaIG-H3 Protein
D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000099222:betaIG-H3 Protein; C508960:TGFB1 protein, human