Reversible differentiation of myofibroblasts by MyoD

Abstract

Myofibroblasts participate in tissue repair processes in diverse mammalian organ systems. The deactivation of myofibroblasts is critical for termination of the reparative response and restoration of tissue structure and function. The current paradigm on normal tissue repair is the apoptotic clearance of terminally differentiated myofibroblasts; while, the accumulation of activated myofibroblasts is associated with progressive human fibrotic disorders. The capacity of myofibroblasts to undergo de-differentiation as a potential mechanism for myofibroblast deactivation has not been examined. In this report, we have uncovered a role for MyoD in the induction of myofibroblast differentiation by transforming growth factor-β1 (TGF-β1). Myofibroblasts demonstrate the capacity for de-differentiation and proliferation by modulation of endogenous levels of MyoD. We propose a model of reciprocal signaling between TGF-β1/ALK5/MyoD and mitogen(s)/ERK-MAPK/CDKs that regulate myofibroblast differentiation and de-differentiation, respectively. Our studies provide the first evidence for MyoD in controlling myofibroblast activation and deactivation. Restricted capacity for de-differentiation of myofibroblasts may underlie the progressive nature of recalcitrant human fibrotic disorders.

Figure 1. TGF-β1 signaling, via the ALK5 receptor, induces stable myofibroblast differentiation

Fibroblasts were maintained in serum-free media for 48 h and subsequently treated with/without TGF-β1 (2 ng/ml) on day 0. (A) A time-course for α-SMA protein expression was determined by Western immunoblotting at the indicated time-points. (B) Cells were incubated with/without media replacement (serum/TGF-β1-free media) at 16 h. Detection of α-SMA was determined by immuno-fluorescence for all conditions at 48 h. (C) Cells were pre-treated with/without pharmacologic inhibitors against ALK5 receptor kinase (SB431542; 0.5µM) 30 m prior to stimulation with/without TGF-β1. α-SMA protein expression was detected by Western immunoblotting on day 2. (D) Media was replaced with TGF-β1-free media with/without the addition pharmacologic inhibitors against ALK5 receptor kinase (SB431542; 0.5µM) and α-SMA protein expression was assessed at the indicated time-points.

Figure 2. MyoD is required for myofibroblast differentiation

(A–B) Fibroblasts were maintained in serum-free media for 48 h and subsequently treated with/without TGF-β1 (2 ng/ml) on day 0. A time-course (0–48 h) of MyoD and α-SMA expression was determined by Western immunoblotting (A), and densitometric analysis (B). (C) Cells were transfected with either a non-targeting control siRNA or using RNAi strategies targeting MyoD (cell line A and B) and stable knockdown was accomplished by selection of transfected cells with geneticin. Cells were then serum starved for 48 h and treated with/without TGF-β1 (2ng/ml) for 48 h. Expression of MyoD and α-SMA was determined by Western immunoblotting.

Figure 3. Differentiated myofibroblasts maintain the capacity for proliferation

Fibroblasts were maintained in serum-free media for 24 h and subsequently treated with/without TGF-β1 (2 ng/ml) for 48 h to induce myofibroblast differentiation (baseline). Cells were then treated with/without 20% serum-containing medium for 72 h. Cell proliferation of was assessed by cell number using a coulter counter (A), BrdU incorporation (B), and immunoflorescence staining with Ki67 (green), α-SMA (red), and DAPI (blue) (C). Bars represent mean ± SEM; n = 4 per group.

Figure 4. Mitogenic signals induce a downregulation of MyoD and α-SMA in myofibroblasts that correlates with increased proliferative capacity

(A–B) TGF-β1-induced myofibroblasts were treated with/without media containing 20% serum for 5 days. Expression of MyoD and α-SMA was determined by Western immuno-blotting (A) and densitometric analysis (B). (C–D) Uuntreated fibroblasts or TGF-β1-induced myofibroblasts were treated with/without media containing 20% serum for 2 d. Serum treated myofibroblasts were trypsinized, re-plated, and treated with/without 20% serum for an additional 5 days. Proliferation was assessed by cell number using a coulter counter (C) and BrdU incorporation (D). (E–F) Cells were transfected with either a non-targeting control siRNA or RNAi targeting MyoD, and treated with/without 20% serum for 48 h. Proliferation was assessed by cell number using a coulter counter (E) and BrdU incorporation (F). Data points on line graphs represent mean ± SEM; n = 3 per group. Bars represent mean ± SEM; n = 4–6 per group.

Figure 5. De-differentiation of myofibroblasts is mediated by the ERK-MAPK pathway that is only partially dependent on MyoD

(A–D) Cells were serum-starved for 24 h, then pre-treated for 30 m with pharmacologic inhibitors indicated, ERK-1/2 (PD98059, 20 µM), p38 (SB203580, 6 µM), and JNK (SP600125, 1 µM) MAPKs, or cyclin-dependent kinases (CDKs; roscovitine, 10 nM) prior to stimulation with PDGF (50 ng/ml) for 48 h. (A–C) Cell lysates were collected and expression of MyoD and α-SMA were determined by Western immunoblotting (A) and densitometric analysis (B–C). (D) Cell were fixed for immunoflorescence staining using antibodies against α-SMA (differentiation marker), Ki67 (proliferation marker), and DAPI (nuclei). Bars represent mean ± SEM; n = 4 per group; *P < 0.5 compared to PDGF alone.

Figure 6. Proposed mechanisms of MyoD in myofibroblast differentiation and dedifferentiation

Fibroblast-to-myofibroblast differentiation is mediated by TGF-β1 ligand-induced activation of the serine-threonine kinase TGF-β type I (ALK5) receptor that signals the induction/activation of MyoD, which then mediates the upregulation of α-SMA that assembles into stress fibers in differentiated myofibroblasts. Myofibroblast dedifferentiation is activated by mitogenic factors such as PDGF that signal via tyrosine kinase receptors which activate the ERK1/2 MAPK and CDKs to downregulate MyoD and α-SMA expression. An independent of effect of ERK1/2 MAPK signaling directly on stress fiber assembly/polymerization may also contribute to myofibroblast dedifferentiation.