A comprehensive analysis of SHP target genes and pathways on hepatic stellate cells and the discovery of a new class of SHP agonists that reduces liver fibrosis

Barbara Renga*, Sabrina Cipriani # , Adriana Carino*, Silvia Marchianò*, Angela Zampella † , Eleonora Distrutti and Stefano Fiorucci*

*Dipartimento di Scienze Chirurgiche e Biomediche, Università degli studi di Perugia Nuova Facoltà di Medicina e Chirurgia, Sant’Andrea delle Fratte, Perugia, Italy #Dipartimento di Medicina, Università degli studi di Perugia Nuova Facoltà di Medicina e Chirurgia, Sant’Andrea delle Fratte, Perugia, Italy †Dipartimento di Farmacia, Università di Napoli Federico II, Napoli, Italy

Background. The small heterodimer partner (SHP) is an orphan receptor that modulates the transcriptional activity of several genes. Because SHP lacks a DNA binding domain, it functions as a negative co-regulator of receptor-dependent signaling pathways. The loss of hepatic SHP is associated with increased expression of profibrogenic markers, accelerated progression of hepatic fibrosis and liver tumor cell invasiveness. We have previously demonstrated that a FXR-SHP regulatory cascade negatively regulates the trans-differentiation and activity of hepatic stellate cells (HSCs), promoting the resolution of liver fibrosis. The exact mechanism by which SHP regulates HSCs is still poorly investigated.Aim of the study. The present study aimed to investigate the expression of SHP target genes and SHP regulated pathways on HSCs as well as the discovery a new class of SHP agonists.

Methods. To investigate the expression of SHP-regulated genes in HSCs we have infected an immortalized rat hepatic stellate cell line (HSC-T6) with a retroviral vector containing the coding sequence of the rat SHP. We have then performed a microarray analysis on both wild type and SHP overexpressing HSC-T6 cells (HSC-T6-SHP) and stimulated with an FXR agonist (i.e.CDCA).Differentially expressed genes (DEGs) were analyzed in HSCT6 andHSCT6 overexpressing SHP. A pathway enrichment analysis was performed using the bioinformatic tools KOBAS; g:profiler and DAVID. The design, synthesis and pharmacological characterization of a novel family of selective SHP ligands based on the isossazol scaffold was also performed.

Results. Up to 574 genes werew differentially expressed (DEGs) in HSC-T6-SHP versus HSC-T6, and 146 genes were modulated by exposure of HSC-T6 to CDCA. The two groups shared 108 genes. After pathway and functional enrichment analysis 46 genes were identified. Some of these genes mainly enriched in the process of HSCs transdifferentiation and activity (i.e. CXCL12, TGFRB2 and RARγ). Validation of microarray results by RT-PCR demonstrated that HSC-T6-SHP had reduced mRNA expression of TGFRB2 in comparison with HSC-T6 cells. In order to discovera novel and selective agonist for SHP, we have synthesized and characterized a new class of chemical compounds based on the isossazol scaffold. The SAR analysis allowed the identification of ISO-COOH as a novel SHP agonist with an EC50 of 9 μM. ISO-COOH was as effective as ATRA in inducing SHP activity in transactivation assay. The treatment of HSC-T6 with ISO-COOH significantly reduced the mRNA expression of TGFRB2 and up-regulation of collagen and αSMA caused by TGFβ1 (p<0.05). ISO-COOH reduced liver fibrosis in the CCL4 model.

Conclusions. The present study suggest a role for SHP in regulating HSCs. Furthermore, the discovery of a novel class of SHP agonists might hold utility in the treatment of hepatic fibrosis.

Figure 3: Molecular structures of compounds 1–7. Transactivation assay on SHP ligand binding domain. HepG2 cells were transiently transfected with the fusion protein SHP/GAL4 and with the reporter vector pGL4.35. 24 hours post-transfection Cells were stimulated with 10 μM or 50 μM compounds 1-7 and with 10 or 50 μM all trans retinoic acid ATRA, used as a positive control. Results are expressed as the mean ± standard error (*p < 0.05 vs not treated cells (NT). Concentration−response curves for compounds 1-2. HepG2 cells transiently transfected with the fusion protein SHP/GAL4 and with the reporter vector pGL4.35 were stimulated with increasing concentrations of compounds 1 and 2 (1, 10 and 50 μM). ATRA (1,

10 and 50 μM) was used as a positive control to evaluate the SHP ligand binding domain activity. (Figure 3, A-D)

Figure5: Effect of compound 2 on AST, Albumin and Bilirubin in mice renedered cirrhotic by administration with CCl4. Hematoxylin and eosin (H&E) staining. Syrius red staining. Image J

quantification of Syrius red staining. C57BL6 mice were treated for 4 weeks with CCl4 alone or in combination with CDCA (5 mg/kg) or compound 2 (ISOCOOH) (30 mg/kg). The relative hepatic

mRNA expression of αSMA, COL1α1, TGFβ1, IL1β, FXR, SHP and BSEP was assayed by Real-Time PCR. Results are the mean ± SE of 6 mice per group. *p<0.05 versus naïve mice. #p<0.05

versus CCl4 alone. (Figure 5, A-E)

Figure 1: Venn diagram of DEGs in HSCT6 stimulated with CDCA and HSCT6 overexpressing SHP. Pathway analysis showing the top enriched canonical pathways. (Figure 1, A-B)

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Figure 2: Scatter plot and clustergram of microarray data showing DEGs that are regulated by SHP (positively in red, negatively in green) in HSC-T6 stimulated with CDCA or HSC-T6 overexpressing SHP. Effect of CDCA treatment and SHP over-expression on the relative mRNA expression of CXCL12, TGFBR2, DAG1 GR, NCoR1 and Rb1. (Figure 2, A-C)

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Figure 4: Pharmacological evaluation on derivative 2(ISOCOOH) in HSC-T6 and HSC-T6 overexpressing SHP. Serum starved HSC-T6 and HSC-T6 overexpressing SHP were stimulated 18 hours with 10 ng/ml TGFβ1 alone

or in combination with 50 μM compound 2. At the end of stimulation the relative mRNA expression of αSMA and COL1α1 was assayed by Real-

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