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Published: 2021-06-18 05:19:19
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Connective tissue growth factor induces collagen I expression in human lung fibroblasts through the Rac1/MLK3/JNK/AP-1 pathway
Many lung inflammatory diseases present pulmonary fibrosis as one of the symptoms, which is characterized by shortness of breath, chronic coughing and discomfort in the chest. It causes modifications of the lung tissues due to overgrowth, hardening and scarring. This occurs mainly due to deposition of collagen, an extracellular matrix (ECM) protein, whose turnover is regulated by fibroblast. Fibroblast is an important cell regulator that is enlisted by a site of injury. This process is driven by release of transforming growth factor-β (TGF-β), platelet-derived growth factor, interleukin-6 (IL-6), and IL-8/CXCL8.
Previously, fibroblast was thought to express no or very low levels of connective tissue growth factor (CTGF). Recent studies, however, indicate that the occurrence of an injury coupled by inflammatory mediators, such as thrombin and TGF-β, induce the CTGF in the fibroblasts. CTGF is responsible for connective tissue repair and formation of new connective tissues by expression of α-smooth muscle actin (α-SMA) and the myofibroblast phenotype. CTGF is also responsible for the formation of the ECM. Type I collagen is one of the most abundantly expressed connective tissue in the body, including lung fibroblasts. It has been discovered that collagen-dependent cellular processes promote the fibroblast proliferation in pulmonary fibrosis. The authors’ previous study had reported that CTGF was activated by thrombin in the lung fibroblasts via an apoptosis signal-regulating kinase 1 (ASK1) /c-Jun N-terminal kinase (JNK) /activator protein-1 (AP-1) dependent pathway. Three other studies have also implicated the CTGF’s involvement in the development of fibrosis via bleomycin-induced collagen expression, scar formation through Janus kinase (JAK) /signal transducer, activation of transcription (STAT) signaling pathway and collaboration with TGF-β in animals. This formed the rationale for the scientists to choose the CTGF as a potential pro-fibrotic marker candidate to detect lung fibrosis.
The research paper evidently shows the association between AP-1, Rac1, JNK and MLK3 with regard to the CTGF and collagen I expression in pulmonary fibrosis. The collagen I gene’s promoter region shows the presence of numerous transcription factor binding sites, including a site for AP-1. In another study, it was confirmed that AP-1 signaling pathway is involved in dermal fibroblast collagen expression. The molecular mechanism of AP-1 stimulated collagen I expression through CTGF is not clear. Rac1 is a GTP-binding (guanosine triphosphate-binding) protein that belongs to the Rho family. It is known to regulate JNK. Under normal conditions Rac1 is inactive by being bound to GDP (guanosine diphosphate). However, when the body is under stress because of an injury, the chemical balance shifts and phosphorylates GDP to GTP, thereby activating Rac1. Activated Rac1 can associate with MLK3, a downstream effector protein. When the phosphorylated Rac1 combines with MLK3, the phosphorylation activates mitogen-activated protein kinases (MAPKs). This MAPK further activates JNK signaling, which induces MAPK-dependent cell multiplication in various cells within the body. Recently, it has been found that a protein binding with Rac1 has a scaffold for the protein Plenty of SH3 (POSH), which forms complexes with JNK and MLK3 to regulate JNK signaling. POSH protein is associated with apoptosis. Rac1 is associated with the AP-1 signaling pathway in renal carcinoma.
Bearing the above-mentioned evidences in mind, the authors of this paper set forth to investigate the role of Rac1 in CTGF induced MLK3/JNK/AP-1 activation pathway and collagen I expression in human lung fibroblasts. They have hypothesized that Rac1 indirectly stimulates CTGF through downstream activation of MLK3/JNK pathway, which further activates c-Jun/AP-1 and leads to enlistment of the collagen I promoter for expression of collagen I in human lung fibroblasts.
The WI-38 human embryonic lung fibroblast cell line was used. The cell line was grown in minimum essential medium (MEM) that contained fetal calf serum (FCS), L-glutamine, non-essential amino acids (NEAAs), sodium pyruvate, penicillin G and streptomycin. The cells were grown in 5% CO2 and 37 °C. For this experimental study, cells between passage number 18 and 30 were used. The cells were studied using cell transfection and immunoblotting, ChIP assay and cell transfection and luciferase assay. For immuneblotting using Western blot, the cells were grown in 6-cm dishes until they reached confluence. After reaching confluence, the cells were lysed with thrombin and with MLK3 inhibitor (K252a), JNK inhibitor (SP600125), and AP-1 inhibitor (curcumin), as necessary. This lysate was subjected to SDS-PAGE and blotted on polyvinylidene difluoride membrane. The blotted membrane was treated with TBST buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.02% Tween 20; pH 7.4). The immunoreactivity of the proteins was visualized and quantified using specific antibodies. Control cells were transfected with pcDNA (plasmid containing cytomegalovirus promoter) plasmid.
ChIP assay was carried out using a commercial kit. The cells were incubated with CTGF and later cross-linked with formaldehyde. The cells were lysed using sonication and centrifuged to separate the supernatant. The supernatant contained the chromatins, which were then immunoprecipitated using specific antibody containing beads. The chromatins were purified, eluted and amplified. PCR was performed to amplify the AP-1 region associated with transcription factor on the collagen I promoter. This step of the experiment helped in mapping the transcription factors.
Collagen I mRNA were amplified in the cells using primers. The cells were then treated with a transcription inhibitor, actinomycin D and then incubated with CTGF to see if the cells followed de novo synthesis of collagen I. It was found that actinomycin D inhibited synthesis of collagen I, thereby proving the fact that collagen is synthesized de novo in response to stimulation by CTGF.
The activity of Rac1 was measured using an assay kit, where the cells were lysed and the list was incubated with PAK1 p21-binding domain agarose beads. Rac1 protein bound to the beads were solubilized and measured using Western blotting via anti-Rac1 antibody derived from mouse monoclonal antibodies. To elucidate the involvement of Rac1, the cells were transfected with the dominant negative mutant Rac1-T17N mutant (RacN17). Such cells overproduced Rac1, but inhibited expression of CTGF to produce collagen I by 81 ± 7%. Thus, the dominant negative mutation in the gene inhibited protein production. This indicated that Rac1 is indeed involved in CTGF-induced collagen I production. This point was validated when the Rac1 activity when measured after the cells’ exposure to CTGF.
The role of JNK in the signaling pathway was determined using SP600125, a JNK inhibitor. SP600125 inhibited collagen I expression by 93 ± 16%. Cells treated with JNK1DN and JNK2DN showed attenuation of gene expression as well. To understand the position of JNK in the signaling pathway, cells were treated with RacN17 and MLK3DN, resulting in inhibition of JNK and consequently, the entire pathway. This indicated that JNK is positioned downstream of both Rac1 and MLK3.
Cell transfection and AP-1 luciferase assay was carried out, where the cells were grown in 12-well plate and transfected with AP-1-Luc and LacZ using Lipofectamine Plus and later co-transfected with one of the dominant negative mutant, RacN17, MLK3DN, JNK1DN, and/or JNK2DN. Plasmids used were expression plasmid-constructs specific for wild type MLK3, MLK3DN, JNK1DN, JNK2DN and pBK-CMVLacZ (LacZ). When all the genes used were dominant negative, it resulted in inhibition of AP-1 luciferase activity. This showed that AP-1 activity is dependent on Rac1, MLK3 and JNK. Further, when an AP-1 inhibitor, curcumin was used, high levels of AP-1 were produced, but the expression of CTGF-induced collagen I production was inhibited. Cells transfected with RacN17 inhibited 66 ± 19%, MLK3DN inhibited 84 ± 16%, JNK1DN inhibited , 58 ± 18% and JNK2DN inhibited 80 ± 9% of the AP-1-luciferase activity. These sets of experiments determined that Rac1, MLK3, JNK and AP-1 did play an important role in inducing CTGF for the production of collagen.
All the transfection experiments, followed by exposure to the various mutant genes and CTGF were concentration dependent and time dependent.
The big picture painted by this research paper was that CTGF is responsible for collagen formation associated with lung fibrosis. This hypothesis has been answered by the siRNA experiment that links fibroblast differentiation and CTGF-induced collagen I expression. Collagen is thought to be a key mediator in implementing CTGF-induced fibrogenic effect. More specifically, the research paper investigated the role of Rac1, MLK3, JNK and AP-1 in activation of CTGF-induced collagen I expression. The research paper succeeded in establishing the fact that all the above mentioned factors had a major role in impacting the activation of CTGF. This was proved by cell transfection experiments that linked the dominant negative mutant version with inhibition of CTGF activation of collagen I production, Rac1 activation assay and AP-1 luciferase assay. The experiments with the dominant negative mutants established the position of each element within the pathway along with establishing their importance. The activity assay determined the level of activity and differences in the activity compared to control. Though the scientists have proved the relation between CTGF, Rac1, MLK3, JNK and AP-1, the actual molecular and biochemical steps within the signal transduction pathway are unclear.

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