TIMP-1 couples RhoK activation to IL-1β-induced astrocyte responses

Abstract

Interleukin-1β (IL-1β) is a pleotropic cytokine known to influence the central nervous system (CNS) responses to injury or infection. IL-1β also directly induces astrocytic expression of tissue inhibitor of metalloproteinases (TIMP)-1, a potent trophic factor and regulator of matrix metalloproteinase activity. In this study, we examined the functional relationship between IL-1β and TIMP-1 and determined that the behavior of astrocytes in response to IL-1β is determined by TIMP-1 expression. Using primary astrocytes from C57Bl/6 mice, we found astrocytes from wildtype (Wt) mice exhibited a robust wound healing response to a scratch wound that was arrested in response to IL-1β. In contrast, TIMP-1 knockout (TIMP-1KO) astrocytes, exhibited minimal response to the scratch wound but an accelerated response following IL-1β-treatment. We also determined that the scratch wound effect in Wt cultures was attenuated by inhibition of Rho kinase but amplified in the TIMP-1KO cultures. We propose that the specific induction of TIMP-1 from astrocytes in response to IL-1β reflects a previously unrecognized physiological relationship where the directionality of astrocytic behavior is determined by the actions of TIMP‑1. These findings may provide additional insight into glial responses in the context of neuropathology where expression of TIMP-1 may vary and astrocytic responses may be impacted by the inflammatory milieu of the CNS.

Keywords: Astrocyte; IL-1β; MMP; ROCK; TIMP-1.

Figure 1. TIMP-1 induction in response to mechanical injury and inflammation

Analysis of TIMP-1 production in primary cultures 0, 4, and 24 hrs in response to control (squares) or IL-1β treatment (triangles) and scratch wound (red lines). 2-way ANOVA, P<0.0002; where ****, P<0.0001 Cntl w/ IL-1β vs. Cntl; **P<0.01 Scratch w/ IL-1β vs. Cntl w/ IL-1β, and * P<0.05, Scratch w/ IL-1β or Scratch vs Cntl.

Figure 2. The physiological effect of IL-1β on astrocytes is inverted in TIMP-1KO cultures

(A). Representative images of WT and TIMP-1KO astrocytes treated with vehicle or IL-1β. (B). Quantitative analysis of scratch recovery (in percent) from baseline scratch wound in confluent Wt and TIMP-1KO astrocyte cultures treated with vehicle or IL-1β (10 ng/ml).ANOVA, P<0.0001; **, P<0.01 TIMP-1KO Cntl vs. Wt Cntl; ***, P<0.001, Wt Cntl vs TIMP-1KO Cntl or Wt IL-1β, and P<0.002 TIMP-1KO IL-1β vs. TIMP-1KO Cntl. Scale bar =

Figure 3. Introduction of recombinant TIMP-1 restores Wt-like phenotype response to TIMP-1KO cultures

(A) Quantification of scratch recovery (in percent from baseline) in TIMP-1KO astrocyte cultures treated with either vehicle or rm-TIMP-1 (10 ng/ml). (B). Quantification of scratch recovery in TIMP-1 KO astrocyte cultures treated with a C-terminal peptide of TIMP-1 [TIMP-1(C); 10 ng/ml]. (C,D) Quantification of scratch recovery in TIMP-1KO and wildtype astrocyte cultures treated broad spectrum MMP-inhibitor, GM6001, or vehicle (control). Student's t test:; ***, P=0.001.

Figure 4. Diminished astrocytic Rho kinase activity in absence of TIMP-1

(A). ROCK activity measured in Wt and TIMP-1KO astrocytes treated with either IL-1β or vehicle (control). Two-way ANOVA; **, P=0.007 for genotype, P<0.018 for treatment effect. (B). Quantification of scratch recovery in Wt and TIMP-1KO astrocyte cultures (in percent from baseline scratch) when treated with either IL-1β or IL-1β and Rho kinase (ROCK) inhibitor. Two-way ANOVA, ** P<0.0009 for genotype interaction, and P<0.01 for IL-1β treatment. (C) Hypothesized IL-1β pathway regulation of RhoK and the influence of TIMP-1 where the absence of receptor-mediated signaling via TIMP-1 (i.e. TIMP-1KO) modifies the physiological response of astrocytes to IL-1β.