TGF-β autoinduction in RPE cells was recently reported as the amplifier of subretinal fibrosis owing to TGF-β–initiated EMT in the pathogenesis of nAMD.
6 In general, TGF-β/TβR–induced EMT requires signaling via SMAD-dependent (canonical) and -independent (noncanonical) pathways, the latter of which includes extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K)/AKT.
12–14 In particular, the activity of SMAD2, the key downstream molecule of TGF-β signaling, is critical for cancer EMT.
15 SMAD signaling was also shown to be involved in proximal tubular epithelial TGF-β autoinduction together with ERK and p38 mitogen-activated protein kinase (MAPK) pathways.
16 In the context of GMT, however, little is known about TGF-β signal transduction and TGF-β autoinduction in Müller glial cells. To explore the possible involvement of TGF-β autoinduction in Müller GMT, we next investigated the impact of TGF-β1/2 application on
TGFB1/2 expression in human Müller glial cells. Stimulation with TGF-β1 and TGF-β2 to Müller cells significantly elevated mRNA levels of both
TGFB1 (
Fig. 2A) and
TGFB2 (
Supplementary Fig. S2A). To determine its downstream intracellular signaling pathways, we pretreated Müller cells with specific inhibitors for ERK1/2, nuclear factor (NF)-κB, PI3K, c-Jun N-terminal kinase (JNK), p38 MAPK, and glycogen synthase kinase (GSK)-3. TGF-β1/2–induced
TGFB1 expression levels were significantly decrease by NF-κB, p38 MAPK, and GSK-3 inhibitors, but enhanced by PI3K inhibitor (
Fig. 2B), which was also true of
TGFB2 expression changes (
Supplementary Fig. S2B). Transfection with small interfering RNA (siRNA)-1 and siRNA-2 against
SMAD2 into Müller cells significantly suppressed
SMAD2 mRNA expression (
Fig. 2C). Silencing of
SMAD2 did not cancel TGF-β1/2–induced expression of
TGFB1 (
Fig. 2D) or
TGFB2 (
Supplementary Fig. S2C), indicating the absence of canonical signaling in Müller glial autoinduction of TGF-β1/2. Consistently, the phosphorylation of NF-κB, p38, and GSK-3α/β increased substantially up to 24 hours after stimulation with TGF-β1 (
Fig. 2E) and TGF-β2 (
Supplementary Fig. S2D). In addition, the inhibition of PI3K alone did not increase
TGFB1 or
TGFB2 expression, but synergistically increased TGF-β1/2–induced
TGFB1 and
TGFB2 expression (
Supplementary Figs. S2E,
S2F). These results indicated that Müller glial autoinduction of TGF-β1/2 was positively regulated via noncanonical pathways by NF-κB, p38 MAPK, and GSK-3, and negatively by PI3K/AKT.