The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

Abstract

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins

“…Furthermore, IPA analysis of the proteomic dataset predicted that the NF-κB pathway, important in pro-inflammatory signaling, was upregulated in RPE cultured in DMEM with 1% sericin. This is in agreement with a previous transcription analysis report by our group in which we demonstrated that sericin conduces pigmentation of RPE by activating the NF-κB pathway [ 8 ]. Despite the higher levels of inflammation-associated proteins, and the predicted activation of the NF-κB pathway in sericin-supplemented RPE cultures, significantly higher cell viability was observed in these cultures compared to cells cultured without sericin.…”

“…These findings are supported by reports proposing sericin-associated superoxide dismutase activity [ 25 , 26 ]. Moreover, through Affymetrix microarray analysis, we recently reported that sericin upregulates superoxide dismutase genes in RPE cultured with sericin [ 8 ]. Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide into oxygen, water and hydrogen peroxide.…”

“…The DHR assay showed a significantly higher level of hydrogen peroxide in RPE cultured with sericin. Interestingly, hydrogen peroxide has been reported to activate NF-κB [ 27 ], and we have shown that increased NF-κB is associated with increased pigmentation in cultured RPE [ 8 ]. In epidermal cells, generation of hydrogen peroxide through ultraviolet radiation is known to cause melanogenesis [ 28 ].…”

“…We have previously reported that the addition of sericin to cell preservation medium improves RPE phenotype following a seven-day preservation period [ 7 ]. We have also shown that sericin has pro-pigmentation effects on cultured RPE, and that these effects are associated with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway [ 8 ].…”

“…Since sericin is considered to be a potent antioxidant [ 5 , 9 ], and antioxidants generally are thought to inhibit NF-κB [ 10 , 11 ], our previous results showing that sericin activates the NF-κB pathway [ 8 ] were unexpected. The present study was designed to follow-up on our previous transcription analysis by investigating global changes at the protein level.…”