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Regulation of RhoA by STAT3 coordinates glial scar formation.

ABSTRACT: Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin-Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3^+/- mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.

SUBMITTER: Renault-Mihara F

PROVIDER: S-EPMC5551705 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Regulation of RhoA by STAT3 coordinates glial scar formation.

Renault-Mihara Francois F Mukaino Masahiko M Shinozaki Munehisa M Kumamaru Hiromi H Kawase Satoshi S Baudoux Matthieu M Ishibashi Toshiki T Kawabata Soya S Nishiyama Yuichiro Y Sugai Keiko K Yasutake Kaori K Okada Seiji S Nakamura Masaya M Okano Hideyuki H

The Journal of cell biology 20170622 8

Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. T ...[more]

PMID: 28642362

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Project description:Astrocytes and microglia play an orchestrated role following spinal cord injury; however, the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood. Herein, microglia were pharmacologically depleted and the effects on the astrocytic response were examined. We further explored the potential mechanisms involving the signal transducers and activators of transcription 3 (STAT3) pathway. For in vivo experiments, we constructed a contusion spinal cord injury model in C57BL/6 mice. To deplete microglia, all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397, starting 2 weeks prior to surgery until they were sacrificed. Cell proliferation was examined by 5-ethynyl-2-deoxyuridine (EdU) and three pivotal inflammatory cytokines were detected by a specific Bio-Plex ProTM Reagent Kit. Locomotor function, neuroinflammation, astrocyte activation and phosphorylated STAT3 (pSTAT3, a maker of activation of STAT3 signaling) levels were determined. For in vitro experiments, a microglia and astrocyte coculture system was established, and the small molecule STA21, which blocks STAT3 activation, was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia. PLX3397 administration disrupted glial scar formation, increased inflammatory spillover, induced diffuse tissue damage and impaired functional recovery after spinal cord injury. Microglial depletion markedly reduced EdU+ proliferating cells, especially proliferating astrocytes at 7 days after spinal cord injury. RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397. Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes. Importantly, in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration. These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation, and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.

Project description:Extracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC's different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain.

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Regulation of RhoA by STAT3 coordinates glial scar formation.

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Regulation of RhoA by STAT3 coordinates glial scar formation.

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