Klotho prevents transforming growth factor-β2-induced senescent-like morphological changes in the retinal pigment epithelium

Degenerative changes of the retinal pigment epithelium (RPE) triggered by transforming growth factor-beta 2 (TGF-beta 2) and oxidative stress play a critical role in the progression of age-related macular degeneration (AMD). The expression of alpha-klotho, an antiaging protein, declines with age, increasing the risk factors for age-related diseases. Here, we investigated the protective effects of soluble alpha-klotho on TGF-beta 2-induced RPE degeneration. The morphological changes induced by TGF-beta 2, including epithelial-mesenchymal transition (EMT), were attenuated in the mouse RPE by the intravitreal injection (IVT) of alpha-klotho. In ARPE19 cells, EMT and morphological alterations by TGF-beta 2 were attenuated by co-incubation with alpha-klotho. TGF-beta 2 decreased miR-200a accompanied by zinc finger e-box binding homeobox1 (ZEB1) upregulation and EMT, all of which were prevented by alpha-klotho co-treatment. Inhibitor of miR-200a mimicked TGF-beta 2-induced morphological changes, which were recovered by ZEP1 silencing, but not by alpha-klotho, implying the upstream regulation of alpha-klotho on miR-200a-ZEP1-EMT axis. alpha-Klotho inhibited receptor binding of TGF-beta 2, Smad2/3 phosphorylation, extracellular signal-regulated protein kinase 1/2 (ERK1/2)-a mechanistic target of rapamycin (mTOR) activation and oxidative stress via NADPH oxidase 4 (NOX4) upregulation. Furthermore, alpha-klotho recovered the TGF-beta 2-induced mitochondrial activation and superoxide generation. Interestingly, TGF-beta 2 upregulated alpha-klotho expression in the RPE cells, and genetic suppression of endogenous alpha-klotho aggravated TGF-beta 2-induced oxidative stress and EMT. Lastly, alpha-klotho abrogated senescence-associated signaling molecules and phenotypes induced by long-term incubation with TGF-beta 2. Hence, our findings indicate that the antiaging alpha-klotho plays a protective role against EMT and degeneration of the RPE, demonstrating the therapeutic potential for age-related retinal diseases, including the dry type of AMD.

Automated summary

Degenerative changes of RPE triggered by TGF-beta 2 and oxidative stress play a critical role in AMD progression. The expression of antiaging protein alpha-klotho declines with age, increasing the risk of age-related diseases. Alpha-klotho protects against TGF-beta 2-induced RPE degeneration and EMT both in vitro and in vivo. It inhibits TGF-beta 2 signaling, EMT, oxidative stress, and senescence-associated signaling.