TGFB1-induced extracellular expression of TGFBIp and inhibition of TGFBIp expression by RNA interference in a human corneal epithelial cell line

Abstract

Purpose: 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.

Figure 1.

TGFB1 induces TGFBIp expression in the HCEC line. Constitutive expression of TGFBIp by uninduced HCECs is shown at 24, 48, and 72 hours after plating. The amount of TGFBIp produced by HCECs is increased up to fourfold (at 24 hours) after the addition of TGFB1 to the media.

Figure 2.

Extracellular TGFBIp production by HCECs is reduced after transfection with siRNAs targeting TGFBI mRNA. Each of the seven commercially available siRNAs (at 30 nM concentration) reduced the amount of extracellular TGFBIp compared with HCECs that were exposed to the transfection reagent but not to an siRNA (no siRNA control). Additionally, the inhibition of TGFBIp production by several of the siRNAs was much greater than that observed with the scrambled siRNA control.

Figure 3.

Selection of the most effective siRNAs targeting TGFBI mRNA and optimization of the concentration of each maximize the inhibition of TGFBIp production by HCECs. Data shown reflect the mean reduction in extracellular TGFBIp production (knockdown) by HCECs after exposure to the optimal concentration of each siRNA compared with the extracellular TGFBIp production by HCECs exposed to the transfection reagent but not to an siRNA (no siRNA control). Experiments were performed three times to obtain a mean knockdown percentage for each siRNA at each of the five concentrations tested.

Figure 4.

Extracellular TGFBIp production by TGFB1-induced HCECs is reduced after transfection with siRNAs targeting TGFBI mRNA. Data shown reflect the mean reduction in extracellular TGFBIp production (knockdown) by HCECs after exposure to the optimal concentration of each siRNA compared with extracellular TGFBIp production by HCECs exposed to the transfection reagent but not to an siRNA (no siRNA control).

Figure 5.

Intracellular TGFBIp production by HCECs is reduced after transfection with siRNAs targeting TGFBI mRNA. Western blot analysis of intracellular TGFBIp and GAPDH protein (internal control) isolated from HCECs 24 hours after transfection with the optimal concentration of four different siRNAs targeting TGFBI mRNA. Also shown is intracellular TGFBIp production by HCECs exposed to the transfection reagent but not to an siRNA (no siRNA control) and pure TGFBIp (external standard).

Figure 6.

TGFBI mRNA expression by HCECs is reduced after transfection with siRNAs targeting TGFBI mRNA. Data shown reflect the mean reduction in TGFBI mRNA expression in HCECs after exposure to the optimal concentration of each siRNA compared with the TGFBI mRNA expression in HCECs exposed to the transfection reagent but not to an siRNA (no siRNA control).