Dual function of TGFβ in lens epithelial cell fate: implications for secondary cataract

Abstract

The most common vision-disrupting complication of cataract surgery is posterior capsule opacification (PCO; secondary cataract). PCO is caused by residual lens cells undergoing one of two very different cell fates: either transdifferentiating into myofibroblasts or maturing into lens fiber cells. Although TGFβ has been strongly implicated in lens cell fibrosis, the factors responsible for the latter process have not been identified. We show here for the first time that TGFβ can induce purified primary lens epithelial cells within the same culture to undergo differentiation into either lens fiber cells or myofibroblasts. Marker analysis confirmed that the two cell phenotypes were mutually exclusive. Blocking the p38 kinase pathway, either with direct inhibitors of the p38 MAP kinase or a small-molecule therapeutic that also inhibits the activation of p38, prevented TGFβ from inducing epithelial-myofibroblast transition and cell migration but did not prevent fiber cell differentiation. Rapamycin had the converse effect, linking MTOR signaling to induction of fiber cell differentiation by TGFβ. In addition to providing novel potential therapeutic strategies for PCO, our findings extend the so-called TGFβ paradox, in which TGFβ can induce two disparate cell fates, to a new epithelial disease state.

FIGURE 1:

TGFβ induces a loss of lens epithelial markers and a gain of EMT/EMyT markers. DCDMLs were cultured for 6 d with or without 4 ng/ml TGFβ1 before fixation and immunostaining for vinculin, the lens epithelial cell markers ZO-1, connexin43, and Pax6, the mesenchymal proteins fibronectin, procollagen 1, and α5 integrin, or the myofibroblast marker αSMA. Note that TGFβ induced a redistribution of vinculin from cell–cell interfaces to focal adhesions, indicative of EMT. Intracellular accumulation of procollagen I is due to low levels of ascorbic acid in the culture medium; supplementation with ascorbic acid stimulated secretion of procollagen I but did not otherwise detectably change the phenotype of myofibroblastic cells in TGFβ-treated DCDMLs (not shown). All markers assessed in a minimum of three independent experiments with similar results.

FIGURE 2:

TGFβ also induces lens fiber cell differentiation. (A, B) DCDMLs were cultured for 6 d with no additions (control), 4 ng/ml TGFβ1, or TGFβ1 plus the TGFβR inhibitor SB-431542 before phase-contrast microscopy (phase) or fixation and immunostaining for proteins exclusive to (AQP0 and the beaded filament proteins CP49/filensin and CP115/phakinin), or highly enriched in (δ−crystallin), differentiating lens fiber cells. In B, some cultures were double stained with the EMT/EMyT marker αSMA or procollagen 1 (procol). Hoechst 33342 was used to detect nuclei. Representative of at least four experiments, except for the SB-431542 data, which are representative of three experiments.

FIGURE 3:

Biochemical assessment of lens fiber and EMT/EMyT marker expression in DCDMLs. DCDMLs were incubated without growth factor (control) or with 4 ng/ml TGFβ1 or 10 ng/ml FGF2 in either the absence or presence of the TGFβR inhibitor SB-431542. After 6 d of culture, cells were assayed for synthesis of the fiber cell differentiation markers δ-crystallin (by [³⁵S]methionine labeling; entire lane shown in Supplemental Figure S1), CP115, and CP49 (by quantitative Western blotting). The cultures were also assayed for expression of the fibrotic markers fibronectin and αSMA by Western blot. (A) Data from a representative experiment. (B) Quantitation of expression of fiber cell differentiation markers, expressed as fold of control DCDMLs cultured with no additions. (C) Quantitation of the effect of SB-431542 on expression of fiber cell markers, expressed as fold of DCDMLs cultured with growth factor alone. Hashtags denote data sets in which SB-431542 completely blocked the ability of TGFβ to up-regulate fiber marker expression in at least three independent experiments. *p ≤ 0.01.

FIGURE 4:

Two different inhibitors of myofibroblast differentiation do not abrogate the ability of TGFβ to induce expression of lens fiber cell markers. DCDMLs were incubated without (control) or with 4 ng/ml TGFβ1 for 6 d in either the absence or presence of vehicle (0.1% DMSO), 200 μM RGDS peptide, or 20 μM GM6001. (A) Cells were fixed and stained with antibodies against procollagen 1 (green) or αSMA (red). Typical of five (GM6001) or three (RGDS) experiments. (B) Cells were lysed and assayed for the EMT/EMyT markers αSMA and fibronectin and the fiber cell differentiation markers δ-crystallin, CP115, and CP49 as in Figure 3. The extent to which each treatment reduced the ability of TGFβ to up-regulate the expression of the indicated protein is graphed relative to TGFβ plus DMSO-only cultures. *p ≤ 0.000.

FIGURE 5:

Inhibitors of p38 and ERK prevent TGFβ from inducing myofibroblast, but not lens fiber cell, differentiation. (A) A 10× stock of either TGFβ or FGF2 in culture medium was added to the growth medium of DCDMLs to reach a final concentration of 4 or 10 ng/ml, respectively. Control cultures received an equal volume of culture medium without growth factor (ctrl), or were left undisturbed (0). Where indicated, cells were pretreated with SB-431542 (SB4) before addition of TGFβ. After a 20-min or 1.5-h incubation, whole-cell lysates were prepared and probed with antibodies specific for the total or phosphorylated (activated) forms of Smad3, p38, or ERK. Fold activation induced by TGFβ over medium-only control (ctrl) is graphed, as is the value obtained in the presence of SB-431542. *p ≤ 0.004. For the remaining data sets, p ≥ 0.2. Representative of three or more independent experiments. Avian species only express ERK2. (B, C) DCDMLs were preincubated with 20 μM SB203580, 15 μM UO126, or vehicle (0.1% DMSO) for 1 h before addition of 4 ng/ml TGFβ, after which cells were cultured in the continuous presence of both drug and TGFβ for 6 d. Cells were processed for either (B) immunofluorescence microscopy (n = 6) or (C) Western blot/metabolic labeling analysis of EMT/EMyT and lens fiber cell markers as in Figure 4. *p ≤ 0.003. For the remaining data sets, p ≤ 0.05.

FIGURE 6:

p38 and ERK activity are not required for TGFβ to induce Smad3-dependent gene expression. DCDMLs were transfected with the SBE4-Luc reporter construct. Where indicated, the cells were preincubated for 1 h with SB-431542, UO126, SB203580, or BIRB 0796 before culture for 2 d with no additions (ctrl) or 4 ng/ml TGFβ1. Expression of luciferase was assessed by Western blot analysis and normalized to β-actin in the same sample. Fold increase expression of reporter by TGFβ over control was 12.7 ± 5.2 (n = 7). The extent to which each treatment inhibited the ability of TGFβ to up-regulate the expression of the reporter is graphed for each condition. *p ≤ 0.000.

FIGURE 7:

FGFR signaling is not essential for induction of fiber cell differentiation by TGFβ. (A) DCDMLs were preincubated with noggin, PD173074, or vehicle (0.1% DMSO) for 1 h before addition of 4 ng/ml TGFβ1. Six days later, cells were processed for Western blot/metabolic labeling analysis of lens fiber cell markers and fibronectin as in Figure 4. *p ≤ 0.000. For the remaining data sets, p ≥ 0.4. (B) Representative Western blot data showing that PD173074 abolishes fiber cell marker expression by FGF but only partially reduces induction by TGFβ. PD173074 also reduces expression of fibronectin by TGFβ.

FIGURE 8:

Inhibitors of MTOR prevent TGFβ from inducing lens fiber cell, but not myofibroblast, differentiation. (A, B) DCDMLs were preincubated with 100 nM rapamycin, 20 μM Ku-0063794, rapamycin + 20 μM SB203580, or vehicle (0.1% DMSO) for 1 h before addition of 4 ng/ml TGFβ or 10 ng/ml FGF2. (A) After 6 d, cells were processed for immunofluorescence staining of AQP0 and αSMA (i–iv) or either AQP0 or CP49 and counterstained with Hoechst 33342 (v–viii). n = 3. (B) After 6 d, cells were processed for Western blot/metabolic labeling analysis of lens fiber cell and EMT/EMyT markers as in Figure 4. (C) A 1-h preincubation with 100 nM rapamycin does not block the ability of a 1.5-h treatment with TGFβ  to activate Smad3 or ERK as assessed by Western blotting (p ≥ 0.46). (D, E) DCDMLs were incubated with KU-0063794, rapamycin, TGFβ1, IGF1, or FGF2 for the indicated period before Western blot assessment of phosphorylation (p) of AKT on serine 473 and total (t) AKT levels. Fold p/t AKT ratio relative to that obtained in DMSO-only controls in the same experiment. *p ≤ 0.02.

FIGURE 9:

TGFβ induces rapamycin-sensitive expression of a reporter of mammalian fiber differentiation in DCDMLs. DCDMLs were transfected with the DCR1-αA promoter-EGFP reporter construct and cultured for 8 d with no additions (control), 10 ng/ml FGF2, or 4 ng/ml FGF2. Where indicated, the cells were incubated with TGFβ in the presence of DMSO (0.1%), SB-431542 (SB4), rapamycin (rap), or SB203580 (SB2). Expression of EGFP was assessed by live-cell imaging of confluent regions of the cultures or by Western blot analysis. Fold increase over untreated control as measured by Western blot is graphed for each condition.

FIGURE 10:

Up-regulation of cell migration by TGFβ in DCDMLs is associated with EMT/EMyT. Confluent DCDMLs grown on a laminin-coated 96-well coverslip were transferred cell side down onto a laminin-coated 48-well plate. The cells were then cultured for 4–6 d with vehicle only (0.1% DMSO; control) or in the continuous presence of either 4 ng/ml TGFβ1 or 5 ng/ml BMP4, with or without inhibitor (SB-431542, SB203580, UO126, or rapamycin) as indicated. Cells migrating from the edge of the coverslip were visualized by phase contrast microscopy (A, C), or after fixation and double staining for α−tubulin (to label all cells) and αSMA (to label lens cells that had undergone EMyT); edge of coverslip indicated by white line (B). Typical of at least three independent experiments.

FIGURE 11:

The multikinase inhibitor rebastinib (DCC-2036) prevents TGFβ from inducing myofibroblast, but not lens fiber cell, differentiation. (A, B) DCDMLs were preincubated with 1 μM rebastinib (DCC) or vehicle (0.1% DMSO) for 1 h before addition of 4 ng/ml TGFβ1, after which cells were cultured in the continuous presence of both drug and TGFβ for 6 d. Cells were processed for (A) immunofluorescence microscopy (n = 4) or (B) Western blot/metabolic labeling analysis of EMT/EMyT and lens fiber cell markers as in Figure 4. The extent to which each treatment affected the ability of TGFβ to up-regulate the expression of the indicated protein is graphed relative to TGFβ plus DMSO-only positive controls. *p < 0.01. (C) Rat lens explants were incubated with 2.5 μM rebastinib for 1 h before a 48-h treatment with or without 4 ng/ml TGFβ2. Fixed explants were stained for α SMA and mounted in medium with DAPI to localize nuclei. For all conditions, n ≥ 8.

FIGURE 12:

Rebastinib prevents TGFβ from activating the p38 pathway but not Smad3. (A) DCDMLs were treated for 3 h with rebastinib (DCC) or vehicle before a 1.5-h incubation with or without 4 ng/ml TGFβ. Whole-cell lysates were prepared and Western blots probed with antibodies specific for the phosphorylated (activated) forms of p38 or Smad3. (B) DCDMLs were treated for 3 h with rebastinib or vehicle before a 30-min incubation with or without the p38 agonist anisomycin (3 μg/ml). Whole-cell lysates were analyzed by Western blotting with antibodies against activated forms of p38 or MKK3/6. (A, B) The percentage inhibition by rebastinib compared with cultures treated with TGFβ + DMSO; for all conditions, n = 4; *p ≤ 0.000. Experiments using TGFβ instead of anisomycin as a p38 agonist were uninformative due to the limited sensitivity of the phospho-MKK3/6 antibody.

FIGURE 13:

A single 1-h treatment with rebastinib has long-term inhibitory effects on p38 activation and induction of myofibroblast differentiation and cell migration by TGFβ. (A–D) On day 1 of culture, DCDMLS were treated with 10 μM rebastinib (DCC) or vehicle (0.1% DMSO) for 1 h, after which the medium was removed and replaced with fresh, drug-free medium. Medium was replaced on days 3 and 5. (A) After 6 d of culture in TGFβ-free medium, cells were incubated with TGFβ1 (4 ng/ml for 1.5 h; n = 3) or anisomycin (3 μg/ml for 30 min; n = 5). Whole-cell lysates were probed with antibodies specific for the phosphorylated (activated) forms of p38 or Smad3. Percentage inhibition of activation of p38 by rebastinib compared with cultures treated with TGFβ + DMSO. *p ≤ 0.000. (B, C) After 6 d of culture in TGFβ-containing medium, cells were processed for immunofluorescence microscopy (B; typical of five experiments) or Western blot/metabolic labeling analysis (C) of EMT/EMyT and lens fiber cell markers. The extent to which rebastinib pretreatment reduced the ability of TGFβ to up-regulate the expression of the indicated protein is graphed relative to cells pretreated with DMSO before addition of TGFβ. *p ≤ 0.001. (D) DCDMLs grown on coverslips were treated with rebastinib or DMSO for 1 h and cultured cell side down for 4 d in the presence of 4 ng/ml TGFβ1 to assess cell migration as in Figure 10. (E) On day 1 of culture, DCDMLs were treated with 25 μM SB203580 (SB2) or 0.1% DMSO for 1 h before drug removal and culture for 6 d with 4 ng/ml TGFβ. Unlike rebastinib (B), short-term treatment with a p38 kinase inhibitor did not block expression of the EMT/EMyT markers αSMA and procollagen 1. The experiment was repeated three times.