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Latest revision as of 15:51, 12 October 2023

Encodes a member of the GATA factor family of zinc finger transcription factors.

GATA4 is a member of the GATA family of zinc finger transcription factor, which regulates gene transcription by binding to GATA elements. GATA4 was originally discovered as a regulator of cardiac development and subsequently identified as a major regulator of adult cardiac hypertrophy. GATA4 is expressed in both embryo and adult cardiomyocytes where it functions as a transcriptional regulator for many cardiac genes, and also regulates hypertrophic growth of the heart. GATA4 promotes cardiac morphogenesis, cardiomyocytes survival, and maintains cardiac function in the adult heart. We aim to use GATA 4 transcription factor to synthesize a fusion protein to enter the cell by endocytosis, in order to suppress the activation of Hepatic stellate cell. Thus, the symptoms consequential with primary chronic liver disease is suppressed by deactivating active HSCs.

Sequence and Features

Usage and Biology

Our project is mainly divided into 3 parts, namely GATA4 fusion protein, liposome carrier and red blood cell "hitchhiker" transportation. We aim to design a part which is synthesised to a fusion protein including the function of suppressing effect of GATA4 transcription factor, and the disagnosis usage of GFP fluoroscence, aiding the target of noticing expression of Therefore, we designed a T7-promoter-based gene expression plasmid carrying GFP coding region When expressed in E. coli BL21 and DH5a cells, this plasmid is expected to produce GFP proteins, which indicates the existance of Hepatic Stellate Cells

Part design

In our project, genetic modified fusion protein serves 2 functions: supressing the activation of HSC^[1], and diagnosis of chronic liver disease at a very primary stage, which usually no specific syptoms and actions are shown. The T7 promotor we added in our modified DNA triggers the expressing of GFP fluoroscence. This part is designed with T7 promoter following with histags and ribosome binding sites.

GFP diagnosis

^[2]When the Nobel Prize in chemistry was awarded for the discovery of green fluorescent protein (GFP) in 2008, the Nobel Committee called GFP a guiding star for biochemistry enabling processes that were previously invisible, such as cancer cells spreading, to be strikingly visible. GFP and its red, blue, yellow, and orange cousins have revolutionized bio-medical research and enabled every major disease, both cause and effect, to “light-up” in the laboratory for researchers to visualize, understand and eventually conquer them. This also applies in our dedicated project but instead of cancer tumour cells, GFP acts as a signalling device for the pedominantly release aSMA receptors from the liver fibrosis site^[3]. When liver fibrosis inducing realease of excessive aSMA receptors is detected, the expression started and GFP is expressed as a signal. In the future, by the advent of GATA4 acting as a "detector", and expressing GFP as a signalling device, it's believed that this fusion protein-driven liposome carrier drug could bring us to a far destination, not only as a quick test to chronic liver disease, but as a beacon of hope in curing chronic liver disease in instant.

GATA4 transciption factor

Myofibroblasts are absent from normal tissues. During normal tissue repair, transient activation of myofibroblasts contributes to restoration of integrity of the tissue by forming a mechanical scar that is usually dissolved when the tissue is repaired. At this stage myofibroblasts are cleared by apoptosis or by inactivation.(1,2) In contrast, persistent myofibroblast activation causes accumulation and contraction of collagenous extracellular matrix (ECM), a condition called “fibrosis.”^[4] By the possible solution of deactivating the activated HSC, our discovery and modified gene shed light on the future of chronic liver disease therapy. Proven that GATA4 transciption factor express effectively accelerate the process of deactivating aHSC, we connected GATA4 isoform and GFP fluoroscence in the well designed plasmid, offering a synthesis of fusion protein for a groundbreaking solution, hasten the progress of liver disease therapy in a breakneck speed. According to the research conducted in 2021^[5], Gata4 overexpression reduces liver fibrosis in vivo, and is proved by a solid-grouned, evidence based series of experiments.

Proof of desired effect of GATA4

To test this hypothesis, ^[6]Universidad Pablo de Olavide researchers evaluated the effect of Gata4 overexpression on liver fibrosis regression in vivo. To this end, mice were treated with CCl4 for 4 weeks to induce liver fibrosis, followed by tail vein injection of Gata4-overexpressing adenovirus (Ad-GATA4) or Ad-GFP as a control. Liver tissues were collected 1 week after adenovirus infection, i.e., 9 days after the last CCl4 injection.(Figure 3)

Figure 3: ^[7] Figure 3.A-C

Histological evaluation using Sirius red revealed diminished fibrillar collagen deposition in the livers of mice injected with Ad-GATA4 compared with both control mice (no infection with adenoviruses) and mice injected with Ad-GFP . Decreased CD45 immunostaining indicated reduced liver inflammation in mice injected with Ad-GATA4 . Of note, injection of Ad-GATA4 in wild-type mice did not have any effect on collagen accumulation or liver inflammation. The repected team evaluated HSC activation in CCl4-treated mice by α-SMA immunohistochemistry. Livers of CCl4-treated mice injected with Ad-GATA4 showed a decreased number of α-SMA–positive areas compared with both control mice (no infection with adenoviruses) and mice injected with Ad-GFP. Quantification of Col1A1 and α-Sma mRNA levels confirmed a reduction of fibrosis and HSC activation in Gata4-overexpressing mice (Figure 4).

Figure 4: ^[8] Figure 3.T-V

Thus, Gata4 overexpression promotes liver fibrosis regression in vivo by deactivating HSCs.

Deactivation of aHSC in practical mean

GATA4 reverts the active phenotype of HSCs by modulating the expression of fibrogenic and antifibrogenic genes.To confirm the cell-autonomous role of GATA4 in HSC deactivation and to get insight into the underlying molecular mechanisms, the team turned to cell culture using LX2 cells, a human HSC cell line that recapitulates many features of the aHSC phenotype. Due to safety consideration and equipment limitations, our team can't conduct cell lysis test on our own. Instead, this citation allows us to solidate our genetic engineering approach on GATA4, in the name of DIMSTAR treatment. The team devised a system that allows robust Gata4 activation in LX2 cells using Ad-GATA4 or Ad-GFP as a control. Adenovirus-mediated overexpression of Gata4 caused a marked reduction in laminin immunoreactivity, which further decreased as a multiplicity of infection (MOI) increased (Figure 5.1).

Figure 5.1: ^[9] Figure 4.B and C

At a MOI of 100, nearly 98% of LX2 cells were transduced. Transduction of LX2 with Ad-GFP at a MOI of 100 did not affect laminin expression (Figure 5.2).

Figure 5.2: ^[10] Figure 4.A

Next, microarray analyses were performed to analyze changes in gene expression in Gata4-overexpressing LX2 cells compared with GFP-overexpressing LX2 cells (Figure 5.3).

Figure 5.3: ^[11] Figure 4.D

Among the most downregulated genes were profibrogenic genes such as ECM components (ACTA2, LAMA1, and COLlA1), metalloprotease inhibitors (TIMP1), TGF-β receptors (TGFβR1 and TGFβR2) and PDGF receptors (PDGFRA and PDGRFB), TLR4, and IL6. Conversely, an increase in the expression of known antifibrogenic factors, such as STAT1, SMAD7, and the transcription factor TCF21, was observed. Quantitative RT-PCR and Western blot analysis confirmed the changes in expression in these fibrosis-related genes (Figure 5.4, 5.5).

Figure 5.4: ^[12] Figure 4.E

Figure 5.5: ^[13] Figure 4.G

Interestingly, these genes showed the reverse expression pattern in the liver tissue of embryos lacking Gata4 specifically in HSCs (10), confirming that GATA4 regulates the expression of multiple genes involved in liver fibrosis (Figure 5.6).

Figure 5.6: ^[14] Figure 4.H and I

References

1. ↑ Arroyo N, Villamayor L, Díaz I, Carmona R, Ramos-Rodríguez M, Muñoz-Chápuli R, Pasquali L, Toscano MG, Martín F, Cano DA, Rojas A. GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells. JCI Insight. 2021 Dec 8;6(23):e150059. doi: 10.1172/jci.insight.150059. PMID: 34699385; PMCID: PMC8675192.
2. ↑ Hoffman RM. Application of GFP imaging in cancer. Lab Invest. 2015 Apr;95(4):432-52. doi: 10.1038/labinvest.2014.154. Epub 2015 Feb 16. PMID: 25686095; PMCID: PMC4383682.
3. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; PMCID: PMC5476301.
4. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; PMCID: PMC5476301.
5. ↑ Arroyo N, Villamayor L, Díaz I, Carmona R, Ramos-Rodríguez M, Muñoz-Chápuli R, Pasquali L, Toscano MG, Martín F, Cano DA, Rojas A. GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells. JCI Insight. 2021 Dec 8;6(23):e150059. doi: 10.1172/jci.insight.150059. PMID: 34699385; PMCID: PMC8675192.
6. ↑ Arroyo N, Villamayor L, Díaz I, Carmona R, Ramos-Rodríguez M, Muñoz-Chápuli R, Pasquali L, Toscano MG, Martín F, Cano DA, Rojas A. GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells. JCI Insight. 2021 Dec 8;6(23):e150059. doi: 10.1172/jci.insight.150059. PMID: 34699385; PMCID: PMC8675192.
7. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.
8. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.
9. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.
10. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.
11. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.
12. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.
13. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.
14. ↑ Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301.