Essential role of matrix metalloproteinases in interleukin-1-induced myofibroblastic activation of hepatic stellate cell in collagen

Abstract

Located within the perisinusoidal space and surrounded by extracellular matrix, hepatic stellate cells (HSC) undergo phenotypic trans-differentiation called "myofibroblastic activation" in liver fibrogenesis. This study investigated the regulation of interleukin-1 (IL-1alpha) on expression of matrix metalloproteinases (MMPs) by HSC grown in three-dimensional extracellular matrix and the role of MMPs in HSC activation. To recapitulate the in vivo "quiescent" state of HSC, the isolated rat HSC were grown in three-dimensional Matrigel or type I collagen. Stimulation with IL-1alpha caused robust induction of pro-MMP-9 (the precursor of matrix metalloproteinase-9) when HSC were cultured in these matrices. IL-1alpha induced a conversion of the pro-MMP-9 to the active form only when the cells were in type I collagen. In collagen lattices, IL-1alpha provoked activation of HSC with induction of MMP-13, MMP-3, and breakdown of the matrix. The HSC activation was completely prevented by a treatment of the cells with tissue inhibitor of metalloproteinase-1 or deprivation of MMP-9. Once fully activated, HSC failed to express MMP-9 and showed attenuated induction of MMP-13 and MMP-3. Further, we demonstrated colocalization of alpha-smooth muscle actin and MMP-9 in a subpopulation of HSC in human fibrotic liver tissues. Thus, this study provides a novel model to enlighten the role of MMPs, particularly that of MMP-9, in HSC activation regulated by a specific cytokine in liver fibrogenesis.

Fig. 1. Induction and activation of MMP-9 during IL-1α-induced activation of HSC in fibrillar type I collagen

Isolated rat HSC were cultured on plastic for 2 days followed by seeding in a 24-well plate either as a monolayer on plastic or embedded into the type I collagen lattice. The monolayer or collagen lattice was stimulated with IL-1α at the indicated concentration. After 6 days of culture, the cells were immunostained for α-SMA (A). Gelatinolytic activities in the conditioned medium were resolved by gelatin zymography (B). Breakdown of the collagen lattice was recorded by a digital camera after removing the soluble fractions (C). The serine proteinase activities were assessed by casein zymography (D). HSC in collagen lattices were stimulated with or without IL-1α for 2 days followed by immunostaining for MMP-9 (E). The gelatinases in conditioned medium from the 6-day culture were enriched by gelatin-conjugated Sepharose 4B followed by Western blot for MMP-9 (F).

Fig. 2. Specific ECM and cytokines are both required for pro-MMP-9 induction and activation by HSC

Isolated quiescent HSC were either plated on plastic as a monolayer or embedded in three-dimensional type I collagen, Matrigel, or fibrin gel as described under “Materials and Methods.” The cells were stimulated with 20 ng/ml TNF-α, 20 ng/ml IL-1α, and 2 ng/ml TGF-βfor 6 days. The gelatinase activities were resolved by zymography.

Fig. 3. IL-1α and type I collagen serve as costimulatory signals for induction of MMP-9 and MMP-13, but collagen is not required for MMP-3 expression

Isolated quiescent HSC were seeded as a monolayer on plastic or embedded in three-dimensional collagen lattice. After treatment for 6 days with the respective cytokines or FBS, the conditioned medium was analyzed for gelatinolytic activities by zymography (A). Conditioned medium was subjected to Western blot analysis for MMP-13 (B). The breakdown of collagen lattices was recorded (C). MMP-3 in the conditioned medium was examined by Western blot (D).

Fig. 4. TIMP-1 inhibits both MMP-9 and activation of HSC in collagen matrix

Isolated quiescent HSC were either plated on plastic as a monolayer or embedded in three-dimensional type I collagen followed by stimulation with or without IL-1α together with TIMP-1 at two concentrations. After 5-day culture the cells were fixed and immunostained for α-SMA. To visualize the intracellular fat droplets in the collagen lattice, photographs were taken under simultaneous visible light and fluorescein isothiocyanate emission (A). The type IV collagenase activities in the conditioned medium were measured by cleavage of fluorogenic peptide and plotted as percentage of the maximal activity (B). The TIMP-1 inhibition of pro-MMP-9 activation was analyzed by gelatinolytic zymography (C). Breakdown of collagen lattices was recorded by photography of the lattices after removal of the soluble fractions (D).

Fig. 5. Depletion of MMP-9 prevents IL-1α-induced activation of HSC in collagen matrix

Isolated quiescent HSC were embedded in type I collagen in the bottom chamber of Transwell. 400 μl of control and gelatin-conjugated Sepharose 4B were loaded into the top chamber. After culture with 20 ng/ml IL-1α for 6 days the cells were stained for α-SMA (A), and conditioned medium was resolved for gelatinase activities (B). The texture of collagen lattices was photographed after removal of medium (C).

Fig. 6. Fully activated myofibroblastic HSC show suppressed cytokine responsiveness for MMP production

HSC were fully activated by a culture on plastic in 10% FBS for 10 days. Myofibroblastic HSC were either plated on plastic as a monolayer or embedded in type I collagen. After culture with or without cytokines for 6 days the conditioned medium was analyzed for gelatinase activities by zymography (A). The conditioned media from quiescent and activated HSC were resolved for MMP-13 by Western blot. The gel lattices after the culture were shown in the lower panel (B). MMP-3 from the fully activated HSC was determined by Western blot (C).

Fig. 7. Colocalization of MMP-9 and α-SMA in human fibrotic livers

Biopsies from two patients with liver fibrosis were examined by dual immunostaining for MMP-9 protein and α-SMA. The anti-MMP-9 polyclonal antibodies, which do not cross-react with other MMPs, were recognized by Cy3-conjugated anti-rabbit IgG. α-SMA was detected by fluorescein isothiocyanate-conjugated anti-mouse IgG. The nucleus was stained by DAPI. Colocalization was visualized by a merger of the two stains.