TGF-ß regulates TGFBIp expression in corneal fibroblasts via miR-21, miR-181a, and Smad signaling.

Transforming growth factor-ß (TGF-ß)-induced gene (TGFBI) protein (TGFBIp) is associated with granular corneal dystrophy type 2 (GCD2). TGFBIp levels can affect GCD2 phenotypes, but the underlying molecular mechanisms have not been fully elucidated. We investigated the involvement of microRNA (miRNA) and TGF-ß in the regulation of TGFBIp expression in corneal fibroblasts. Ectopic expression of miR-9, miR-21, and miR-181a significantly decreased TGFBIp levels. Conversely, expression of miR-21 and miR-181a was induced by TGF-ß1. Expression of miR-21 was 10-fold higher than that of miR-9 and miR-181a in corneal fibroblasts. Additionally, TGF-ß1 expression was significantly higher than that of TGF-ß2 and TGF-ß3 in corneal fibroblasts, whereas expression of all three TGF-ß forms was not significantly different between wild-type (WT) and GCD2 homozygotes (HO) corneal fibroblasts. Taken together, these data indicate that TGFBIp expression is positively regulated by TGF-ß, whereas TGF-ß-induced miR-21 and miR-181a negatively regulate TGFBIp expression. In conclusion, TGFBIp levels in corneal fibroblasts are controlled via the coordinated activity of miR-21 and miR-181a and by Smad signaling. Pharmacologic modulation of these miRNAs and TGF-ß signaling could have therapeutic potential for TGFBI-associated corneal dystrophy, including GCD2.

Melatonin induces autophagy via an mTOR-dependent pathway and enhances clearance of mutant-TGFBIp.

The hallmark of granular corneal dystrophy type 2 (GCD2) is the deposit of mutant transforming growth factor-ß (TGF-ß)-induced protein (TGFBIp) in the cornea. We have recently shown that there is a delay in autophagic degradation of mutant-TGFBIp via impaired autophagic flux in GCD2 corneal fibroblasts. We hypothesized that melatonin can specifically induce autophagy and consequently eliminate mutant-TGFBIp in GCD corneal fibroblasts. Our results show that melatonin activates autophagy in both wild-type (WT) and GCD2-homozygous (HO) corneal fibroblast cell lines via the mammalian target of rapamycin (mTOR)-dependent pathway. Melatonin treatment also led to increased levels of beclin 1, which is involved in autophagosome formation and maturation. Furthermore, melatonin significantly reduced the amounts of mutant- and WT-TGFBIp. Treatment with melatonin counteracted the autophagy-inhibitory effects of bafilomycin A1, a potent inhibitor of autophagic flux, demonstrating that melatonin enhances activation of autophagy and increases degradation of TGFBIp. Cotreatment with melatonin and rapamycin, an autophagy inducer, had an additive effect on mutant-TGFBIp clearance compared to treatment with either drug alone. Treatment with the selective melatonin receptor antagonist luzindole did not block melatonin-induced autophagy. Given its ability to activate autophagy, melatonin is a potential therapeutic agent for GCD2.

Uncoupling by (--)-epigallocatechin-3-gallate of ATP-sensitive potassium channels from phosphatidylinositol polyphosphates and ATP.

Of green tea catechins, (--)-epigallocatechin-3-gallate (EGCG) and (--)-epicatechin-3-gallate (ECG), but not (--)-epicatechin and (--)-epigallocatechin, inhibit the activity of ATP-sensitive potassium (K(ATP)) channels at tens of micromolar concentrations, ECG being three times more effective than EGCG. Further, we found that by using cloned beta cell-type K(ATP) channels, only EGCG at 1 microM, a readily achievable plasma concentration by oral intake in humans, but not other epicatechins, significantly blocked channel reactivation after ATP wash-out, suggesting that interaction of phosphatidylinositol polyphosphates (PIP) with the channel was impaired by EGCG. In addition, a 10-fold higher concentration of EGCG reduced the channel sensitivity to ATP, but not AMP and ADP. This effect of EGCG was greater in the channel with the sulfonylurea receptor (SUR) than with the inwardly rectifying K(+) channel (Kir6.2) alone. Neomycin, a polycation, profoundly suppressed the effect of EGCG. Expectedly, glucose-stimulated cytosolic Ca(2+) elevation in rat pancreatic beta cells, and insulin secretory responses to high glucose loading in vivo were impaired by EGCG. In rabbit cardiac myocytes, dinitrophenol-induced opening of the channel was delayed by 1 microM EGCG. These results suggest that EGCG may interact with PIP-binding sites on the Kir6.2 subunit. SUR further endows EGCG with an ability to interfere with an interaction of the gamma-phosphate tail of ATP with Kir6.2. The specificity of EGCG possibly implies that 5'-OH of the B-ring on the pyrogallol moiety in the EGCG molecule may be critical for these actions of EGCG on the K(ATP) channel.