Part 2:Anti-fibrotic Effect Of 6-Bromo-indirubin-3 ′ -oxime (6-BIO) Via Regulation Of Activator Protein-1 (AP-1) And Specificity Protein-1 (SP-1) Transcription Factors in Kidney Cells

For Part 1, click here.

3.3. 6-BIO attenuates fibrosis markers and fibrosis-associated signaling pathways in the proximal tubule (HK-2) and interstitial fibroblast (NRK49F) cells

Next, we confirmed the effect of 6-BIO on proximal tubular cells and interstitial fibroblast cells. We observed a concentration- and time-dependent reduction of GSK3β mRNA expression by 6-BIO in HK-2 and NRK49F cells (Fig. S2A and B), and the expression of ECM and EMT-related proteins involved in kidney cell fibrosis was also reduced (Fig. S2E). Moreover, enhanced expression of GSK3β, upon TGFβ exposure of these cells, also decreased by 6-BIO treatment (Fig. S2C and D). Fig. 3 shows protein expression of kidney fibrosis markers in HK-2 and NRK49F cells treated with TGFβ. As shown in Fig. 3A, the expression of PAI-1 and CTGF proteins increased significantly in response to treatment with 5 and 10 ng/mL TGFβ in HK-2 and NRK49F cells, respectively. However, their expression reduced remarkably after treatment with 6-BIO (Fig. 3B). SMAD and MAPK signaling pathways were upregulated by TGFβ treatment, which was reversed by the 6-BIO treatment (Fig. 4). Next, we investigated the expression of AP-1 and SP-1 transcription factors involved in TGFβ-mediated signaling in HK-2 and NRK49F cells. Fig. 4D shows that the TGFβ−mediated increase in P-C-JUN and P-C-FOS was reduced in response to treatment with 6-BIO and JNK inhibitor SP600125 and upon dominant-negative C-JUN transfection. Fig. 4E shows the change in protein intensity relative to control. Moreover, TGFβ-mediated increase in SP-1 level was significantly attenuated in response to treatment with 6-BIO and SP-1 siRNA (Fig. 4F). Fig. 4G shows the change in protein intensity relative to control. These results suggested that TGFβ/SMAD and MAPK signaling as well as transcription factors, AP-1, and SP-1, are regulated by 6-BIO.

3.4. 6-BIO attenuates nuclear expression of P-C-JUN, P-C-FOS, and SP-1 proteins in proximal tubular and interstitial fibroblasts cells

Fig. 5A and B show the localization of PAI-1 and CTGF with P-C-JUN, P-C-FOS, and SP-1. Red staining of P-C-JUN, P-C-FOS, and SP-1 indicated their overexpression in the nucleus following TGFβ treatment; similarly, green staining of PAI-1 and CTGF indicated their over-expression in the cytosol. However, the expression levels of P-C-JUN, P-C-FOS, and SP-1 were reduced by the 6-BIO treatment. These results suggested that 6-BIO directly affects AP-1 and SP-1, the transcription factors regulating PAI-1 and CTGF expression.

3.5. 6-BIO attenuates PAI-1, CTGF, AP-1, and SP-1 promoter activity in proximal tubular and interstitial fibroblasts cells

Next, we investigated whether 6-BIO inhibits the promoter activities of PAI-1, CTGF, AP-1, and SP-1 genes. The promoter activity of PAI-1 and CTGF increased in response to TGFβ but was reduced by the 6-BIO treatment (Fig. 6A and B). Moreover, the decrease was notable when cells were treated with 6-BIO alone than in the presence of TGFβ. AP-1 and SP-1 bind to the promoters of PAI-1 and CTGF. Fig. 6C and 6D show the promoter activity of AP-1 and SP-1. AP-1 and SP-1 luciferase activity was increased by TGFβ treatment and decreased by 6-BIO in a dose-dependent manner. Fig. 6E shows the expression of the transcription factor in nuclear protein extracts. Thus, we confirmed that the nuclear expression of P-C-JUN, P-C-FOS, and SP-1 increased in response to TGFβ treatment and was suppressed by 6-BIO treatment. Fig. 6F shows the change in protein intensity relative to control. In addition, our results suggest that 6-BIO acts on the transcription factor binding site of the promoter to weaken the binding of AP-1 and SP-1. We performed in vitro promoter binding assay in the presence of 6-BIO to confirm the same. The binding of nuclear proteins and P-C-JUN, P-C-FOS, and SP-1 was weakened by the addition of 6-BIO in a concentration-dependent manner (supplementary Fig. 3). These results indicate that 6-BIO inhibits PAI-1 and CTGF promoter activities as well as AP-1 and SP-1 functions; thus, it may have therapeutic potential in kidney fibrosis.

4. Discussion

Using the rat model of UUO and TGFβ-treated kidney proximal tubules derived and interstitial fibroblast cell lines, we demonstrated that 6-BIO treatment can attenuate kidney damage. The UUO model is a representative animal model of obstructive nephropathy that is characterized by progressive tubulointerstitial fibrosis [43]. Histopathological analysis of kidney tissues obtained from the UUO model showed that the ECM accumulation and tubular atrophy in the interstitial space were restored by the 6-BIO treatment. In addition, using TGFβ-treated kidney proximal tubule-derived and interstitial fibroblast cells, we demonstrated that 6-BIO treatment can attenuate kidney damage. In the UUO model and TGFβ-treated HK-2 and NRK49F cells, 6-BIO inhibited the expression of kidney fibrosis markers as well as MAPK and SMAD signaling pathways. In addition, 6-BIO treatment reduced the expression of P-C-JUN, P-C-FOS, and SP-1 proteins induced by UUO and TGFβ, as well as the nuclear expression and promoter activity of AP-1 and SP-1 in TGF-β-treated HK-2 and NRK49F cells. Inhibition of AP-1 and SP-1 transcription factors by 6-BIO treatment has been associated with the inhibition of ECM accumulation that leads to exacerbation of kidney fibrosis. This is achieved by suppressing MAPK and SMAD signaling pathways that lead to a reduction in the expression of PAI-1 and CTGF. Our results suggest that 6-BIO may be a potential therapeutic agent against kidney diseases, as well as for preventing kidney damage caused by UUO and TGFβ.

Click to organic Cistanche for kidney disease

The semisynthetic cell-permeable indirubin derivative 6-BIO belongs to a bis-indole family and naturally occurs in edible gastropod mollusks and plants [12]. Indirubin contains an intramolecular hydrogen bond between the 2-C double bond O and the N1-H group and another intermolecular hydrogen bond between the molecules. 6-BIO has a chemically modified structure in which the 6-position is substituted with Br to overcome the disadvantage of poor pharmacokinetic properties due to the low water solubility of indirubin [44]. Many indirubins, because of competitive binding with ATP, act as dual inhibitors of cyclin-dependent kinase (CDK) and glycogen synthase kinase-3β (GSK-3β), exhibiting more than 16-fold selectivity over CDK5 [12]. In addition, 6-BIO exhibits markedly selective inhibition of GSK-3β with an IC50 value of 0.005 μM [12]. According to reports by several groups, 6-BIO completely inhibits MCF-7 cell proliferation and blocks migration by approximately 75% at a concentration of 10 μM, and inhibits phosphorylation of PDK1. This showed that 6-BIO was inserted into the binding pocket of PDK1 to form three hydrogen bonds with residues in the hinge region, and the binding pocket readily accommodated the additional Br atoms in 6-BIO [45]. With these characteristics, 6-BIO is being studied as a new drug to regulate cellular processes such as aging-related inflammation, oxidative stress, cell survival, proliferation, and apoptosis in cancer, diabetes, and degenerative diseases [46,47]. However, studies on gene regulation and molecular mechanisms related to kidney cell fibrosis are scarce. Therefore, we sought to investigate whether 6-BIO attenuated kidney fibrosis by inhibiting PAI-1 and CTGF gene regulation in kidney cells, thereby reducing ECM accumulation.

Several studies have used the UUO model to report a correlation between ECM accumulation and chronic kidney disease caused by tubular interstitial fibrosis [43]. Moreover, overexpressed PAI-1 and CTGF are reportedly associated with high ECM accumulation during the progression of chronic kidney disease. Thus, identifying a therapeutic molecule that inhibits the signaling and transcription of genes associated with enhanced ECM accumulation is important for effectively inhibiting kidney fibrosis. Therefore, in this study, we evaluated 6-BIO, one of the inhibitors of glycogen synthase kinase 3β (GSK-3β), in kidney fibrosis.

We studied the effect of PAI-1 and CTGF proteins on kidney cell fibrosis and confirmed that their expression was regulated by common transcription factors, AP-1, and SP-1. The transcription factor, AP-1, is a heterodimer composed of proteins belonging to the JUN (C-JUN, JUNB, and JUND), FOS (C-FOS, FOSB, Fra-1, and Fra-2), ATF (activation transcription factor), and JDP (JUN dimerization protein) family [48]. It regulates several cellular processes such as cell proliferation, death,

differentiation, and vascularization. An earlier study reported that AP-1 activity is increased in the liver and lungs by the activation of type VI collagen and fibroblasts [49,50]. The two main components of AP-1, C-JUN, and C-FOS respond to a variety of stimuli, such as cytokines, growth factors, and stress [51]. We observed increased phosphorylation of C-JUN and C-FOS in a UUO rat model and TGFβ-treated HK-2 and NRK49F cells (Figs. 2D and 4D). C-JUN and C-FOS form heterodimers and bind to the AP-1 binding site in DNA, thereby acting as transcription factors to regulate the expression of other genes. In addition, phosphorylation of ERK1/2 and JNK activates C-FOS and C-JUN, respectively. In our study, the phosphorylation of ERK1/2 and JNK, as well as C-JUN and C-FOS in the nucleus was remarkably increased in response to treatment with TGFβ. However, 6-BIO treatment significantly reversed these effects. Our study showed that 6-BIO efficiently inhibited the phosphorylation of ERK1/2 and JNK and decreased the activity of transcription factor AP-1, thereby inhibiting the phosphorylation of C-JUN and C-FOS which are its downstream effectors. SP-1 inhibition in hepatic astrocytes exerts an anti-fibrotic effect [52]. Another study showed that SP-1 played important role in fibrosis [52,53]. Therefore, we evaluated whether 6-BIO could inhibit the transcription factor SP-1. We observed that the expression of SP-1 protein increased in response to TGFβ treatment as well as in the UUO model, whereas it decreased following 6-BIO treatment. Moreover, the nuclear expression of SP-1 induced by TGFβ was suppressed by the 6-BIO treatment. As a result, promoter activities of PAI-1 and CTGF, which have AP-1 and SP-1 binding sites, were also reduced by treatment with 6-BIO (Fig. 6). Therefore, these findings suggest that the decrease in promoter activity of PAI-1 and CTGF by 6-BIO treatment lowered the PAI-1 and CTGF protein expression, and decreased ECM accumulation, thereby reducing kidney cell fibrosis.

5. Conclusion

6-BIO treatment decreased PAI-1 and CTGF expression in the UUO model and TGFβ treated kidney cells. In addition, 6-BIO inhibited the TGFβ-induced activation of SMAD and MAPK signaling, while also inhibiting the activities of AP-1 and SP-1, two of the several transcription factors binding to the promoters of PAI-1 and CTGF genes. Inhibition of AP-1 and SP-1 appears to be one of the key mechanisms by which 6-BIO inhibits kidney fibrosis. Thus, our results suggest that 6-BIO may be a potential therapeutic agent against kidney diseases.

CRediT authorship contribution statement

Jung Sun Park: Conceptualization, Methodology, Data curation, Writing − original draft preparation, Writing − review & editing, Visualization, Funding acquisition. In Ae Jung: Data curation, Writing − review & editing. Hong Sang Choi: Data curation, Writing − review & editing. Dong-Hyun Kim: Methodology, Data curation. Hoon In Choi: Methodology, Data curation. Eun Hui Bae: Writing − review & editing, Supervision. Seong Kwon Ma: Writing − review & editing,

6. References

The source is from Jung Sun Park et, al on Biomedicine & Pharmacotherapy 145 (2022) 112402