RhoA Ambivalently Controls Prominent Myofibroblast Characteritics by Involving Distinct Signaling Routes

Jatho, Aline; Hartmann, Svenja; Kittana, Naim; Mügge, Felicitas; Wuertz, Christina M.; Tiburcy, Malte; Zimmermann, Wolfram‐Hubertus; Katschinski, Dörthe M.; Lutz, Susanne

doi:10.1371/journal.pone.0137519

Cited by 17 publications

(22 citation statements)

References 43 publications

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“…This is in line with the previous observations that RhoA is a key regulator of the mechanical properties of fibroblasts (31)(32)(33). These mechanical changes in the RhoA-KO fibroblasts were linked to the loss of wide stress fibers and large focal adhesions.…”

Section: Discussionsupporting

confidence: 92%

RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo

Alkasalias

Alexeyenko

Hennig

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins overexpressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumorinhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of α-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.Rho GTPases | RhoA | cancer-associated fibroblasts | tumor-inhibitory capacity | cytoskeleton

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Section: Discussionsupporting

confidence: 92%

RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo

Alkasalias

Alexeyenko

Hennig

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…This allowed us to compare the effects of Fasudil and H1152P, to demonstrate the concentration-dependent effect of SR-3677, to confirm that ROCKs play a role downstream of TGF-β in cardiac fibroblasts behaviour as suggested before [10], and to validate the importance of ROCKdependent signalling events like the actin-MRTF-LOX regulation. In addition to the presented pharmacological study we have demonstrated in the past that ECT can be prepared from male and female cells to study sex-differences in cardiac fibrosis and from virally transduced cells allowing loss-and gain-of function studies [15,18,30,42].…”

Section: Discussionmentioning

confidence: 99%

“…In vitro, ROCKs regulate fibrosis-associated genes, including connective tissue growth factor (CTGF), fibroblast growth factor 2 (FGF2), α-smooth muscle actin (SMA) and pro-collagen I [10,[13][14][15]. ROCKs are also implicated in cardiac fibroblast migration and proliferation [16][17][18]. All in vitro studies have been carried out using purified cardiac fibroblasts in 2D cultures, and the effects of ROCK inhibition on the behaviour of cardiac fibroblasts embedded in a more physiological 3D environment have not been investigated.…”

Section: Introductionmentioning

confidence: 99%

“…We perform linear ultimate destructive tensile measurements in order to obtain information on tissue stiffness (Young's modulus, slope of the elastic region of the stress-strain curve) and on the strain to failure point reflecting the maximal extensibility of the tissue. We have previously demonstrated that this model is suitable to perform gain and loss of function experiments as well as small molecule studies [15,18,30].…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of Rho-associated kinases suppresses cardiac myofibroblast function in engineered connective and heart muscle tissues

Santos

Hartmann

Zimmermann

et al. 2019

Journal of Molecular and Cellular Cardiology

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Cardiac fibrosis is a hallmark of heart failure for which there is no effective pharmacological therapy. By genetic modification and in vivo inhibitor approaches it was suggested that the Rho-associated kinases (ROCK1 and ROCK2) are involved in pro-fibrotic signalling in cardiac fibroblasts and that they may serve as targets for anti-fibrotic therapies. We demonstrate that simultaneous inhibition of ROCK1 and ROCK2 strongly interfered with tissue formation and their biomechanical properties in a model of engineered connective tissue (ECT), comprised of cardiac fibroblasts and collagen. These effects were observed with both rat and human ECT. Inhibitors of different chemistries, including the isoquinoline inhibitors Fasudil and H1152P as well as the pyrazol-phenyl inhibitor SR-3677, showed comparable effects. By combined treatment of ECT with TGF-β and H1152P, we could identify ROCK as a mediator of TGF-β-dependent tissue stiffening. Moreover, expression analyses suggested that lysyl oxidase (LOX) is a downstream target of the ROCK-actin-MRTF/SRF pathway and inhibition of this pathway by Latrunculin A and CCG-203971 showed similar anti-fibrotic effects in the ECT model as ROCK inhibitors. In line with the collagen crosslinking function of LOX, its inhibition by βaminopropionitrile resulted in reduced ECT stiffness, but let tissue compaction unaffected. Finally, we show that ROCK inhibition also reduced the compaction and stiffness of engineered heart muscle tissues. Our results indicate that pharmacological inhibition of ROCK has a strong anti-fibrotic potential which is in part due to a decrease in the expression of the collagen crosslinking enzyme lysyl oxidase.

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“…Primary rat tail and cardiac fibroblasts were derived from neonatal rats (day 1–3) and cultivated as described earlier …”

Section: Methodsmentioning

confidence: 99%

Hypoxia‐stimulated membrane trafficking requires T‐plastin

et al. 2017

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RhoA Ambivalently Controls Prominent Myofibroblast Characteritics by Involving Distinct Signaling Routes

Cited by 17 publications

References 43 publications

RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo

RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo

Inhibition of Rho-associated kinases suppresses cardiac myofibroblast function in engineered connective and heart muscle tissues

Hypoxia‐stimulated membrane trafficking requires T‐plastin

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