Hydroxyflutamide affects connexin 43 via the activation of PI3K/Akt-dependent pathway but has no effect on the crosstalk between PI3K/Akt and ERK1/2 pathways at the Raf-1 kinase level in primary rat Sertoli cells

Chojnacka, Katarzyna; Zarzycka, Marta; Hejmej, Anna; Mruk, Dolores D.; Gorowska, E.; Kotula–Balak, Małgorzata; Klimek, Monika; Bilińska, Barbara

doi:10.1016/j.tiv.2015.09.027

Cited by 22 publications

(12 citation statements)

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“…The proteins were extracted from testicular tissue (n = 6/each group) with a cold RIPA buffer (Thermo Scientific) supplemented with protease inhibitors (Sigma-Aldrich). Separation of protein preparations by SDS-PAGE under reducing conditions and transfer of proteins to polyvinylidene difluoride membranes were performed as described before [28]. Nonspecific binding sites were blocked with a solution of 5% (wt/v) non-fat dry milk containing 0.1% (v/v) Tween® 20.…”

Section: Western Blot Analysismentioning

confidence: 99%

Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium

Kamińska

Marek

Pardyak

et al. 2020

Reprod Biol Endocrinol

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Background: Onset of spermatogenesis at puberty is critically dependent on the activity of hypothalamic-pituitarygonadal axis and testosterone production by Leydig cells. The aim of this study was to examine whether activation of Notch receptors and expression of Notch ligands and effector genes in rat seminiferous epithelium are controlled by androgen signaling during puberty. Methods: Peripubertal (5-week-old) Wistar rats received injections of flutamide (50 mg/kg bw) daily for 7 days to reduce androgen receptor (AR) signaling or a single injection of ethanedimethane sulphonate (EDS; 75 mg/kg bw) to reduce testosterone production. Gene and protein expressions were analyzed by real-time RT-PCR and western blotting, respectively, protein distribution by immunohistochemistry, and steroid hormone concentrations by enzymelinked immunosorbent assay. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test or by Kruskal-Wallis test, followed by Dunn's test. Results: In both experimental models changes of a similar nature in the expression of Notch pathway components were found. Androgen deprivation caused the reduction of mRNA and protein expression of DLL4 ligand, activated forms of Notch1 and Notch2 receptors and HES1 and HEY1 effector genes (p < 0.05, p < 0.01, p < 0.001). In contrast, DLL1, JAG1 and HES5 expressions increased in seminiferous epithelium of both flutamide and EDS-treated rats (p < 0.05, p < 0.01, p < 0.001). Conclusions: Androgens and androgen receptor signaling may be considered as factors regulating Notch pathway activity and the expression of Hes and Hey genes in rat seminiferous epithelium during pubertal development. Further studies should focus on functional significance of androgen-Notch signaling cross-talk in the initiation and maintenance of spermatogenesis.

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Section: Western Blot Analysismentioning

confidence: 99%

Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium

Kamińska

Marek

Pardyak

et al. 2020

Reprod Biol Endocrinol

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“…The protein concentration was estimated by the Bio-Rad DC Protein Assay Kit with BSA as a standard (Bio-Rad Laboratories). Samples containing 20 lg protein were resolved by SDS-PAGE under reducing conditions, transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA), and analyzed by Western blotting with the respective primary antibodies as previously reported (Chojnacka et al, 2016). Briefly, non-specific binding sites were blocked with a solution of 5% (wt/v) non-fat dry milk containing 0.1% (v/v) Tween â 20.…”

Section: Western Blot Analysismentioning

confidence: 99%

Notch signaling regulates nuclear androgen receptor AR and membrane androgen receptor ZIP9 in mouse Sertoli cells

et al. 2019

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Background Notch signaling pathway is involved in contact‐dependent communication between the cells of seminiferous epithelium, and its proper activity is important for undisturbed spermatogenesis. Objectives The aim was to assess the effect of Notch pathway inhibition on the expression of nuclear (AR) and membrane (ZIP9) androgen receptors and androgen‐regulated genes, claudin‐5 and claudin‐11, in TM4 mouse Sertoli cell line. Materials and methods DAPT (γ‐secretase inhibitor) treatment and recombination signal binding protein silencing were employed to reduce Notch signaling, whereas immobilized ligands were used to activate Notch pathway in TM4 cells. To reveal specific effect of each androgen receptor, AR or ZIP9 silencing was performed. Results Notch pathway inhibition increased the expression of AR and ZIP9 mRNA and proteins (p < 0.01; p < 0.05) in TM4 cells, whereas incubation with Notch ligands, rDLL1 or rJAG1, reduced AR (p < 0.01; p < 0.001) and ZIP9 (p < 0.05; p < 0.01) expressions, respectively. Testosterone enhanced the expression of both receptors (p < 0.05; p < 0.01). Androgen‐regulated claudin‐5 and claudin‐11 (p < 0.01; p < 0.001) and cAMP (p < 0.001) were elevated in Notch‐inhibited cells, while activation of Notch signaling by DLL1 or JAG1 reduced claudin‐11 or claudin‐5 level (p < 0.01; p < 0.001), respectively. Discussion Our findings indicate opposite effect of Notch and androgen signaling on the expression of androgen receptors in TM4 cells. We demonstrated that AR expression is regulated by DLL1‐mediated Notch signaling, whereas JAG1 is involved in the regulation of ZIP9. The expression of both claudins and cAMP production is under inhibitory influence of Notch pathway. The effects of Notch signaling on claudin‐5 and claudin‐11 expression are mediated by ZIP9 and AR, respectively. Conclusion Notch signaling may be considered as an important pathway controlling Sertoli cell physiology, and its alterations may contribute to disturbed response of Sertoli cells to androgens.

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“…However, some reports have shown a negative correlation between Cx43 and Akt activation, caused by direct interactions between Cx43 and Akt in glioblastoma (15). In addition, activation of the PI3K-Akt pathway by 2-hydroxyflutamide resulted in reduced levels of Cx43 in rat Sertoli cells (18). Immunoprecipitation analysis (Figure 3 and Figure S5) showed an interaction between Cx43 and Akt in parental cells treated with resveratrol or with a combination of cetuximab and resveratrol.…”

Section: Cx43 Interacts With Akt In Resveratrol-treated Parental Cellsmentioning

confidence: 91%

“…For example, Cx43 inhibits p-Akt through proteinprotein interactions in glioblastoma (15) but contributes to p-Akt in cardiomyocytes (16). p-Akt also upregulates (17) or downregulates (18) the phosphorylation of Cx43 (p-Cx43), depending on the cell type. Phosphorylation is a necessary step for Cx43 to form gap junctions (19), which sensitizes cancer cells to many antitumor drugs (20).…”

Section: Introductionmentioning

confidence: 99%

Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

Wang

Wang²,

Wu³

et al. 2020

Front. Oncol.

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Cetuximab is a monoclonal antibody that acts as an anti-epidermal growth factor receptor (EGFR) agent. Cetuximab inhibits the phosphorylation and activation of EGFR and blocks downstream signal pathways of EGF/EGFR, including Ras-Raf-MAPK and PI3K-Akt pathways. Akt activation is an important factor in cetuximab resistance. It has been reported that resveratrol and connexin 43 regulate Akt in different ways based on tissue type. Since connexin 43 interacts with Akt, and resveratrol is known to upregulate connexin 43, we investigated whether resveratrol can sensitize colorectal cancer cells to cetuximab via connexin 43 upregulation. Our work confirmed that resveratrol increases the inhibition of growth by cetuximab in vitro and in vivo, upregulates connexin 43 expression and phosphorylation, increases gap junction function, and inhibits the activation of Akt and NFκB in parental or cetuximab-treated parental HCT116 and CT26 cells. Resveratrol did not exhibit these effects on connexin 43-shRNA transfected cells, so connexin 43 upregulation may contribute to Akt inhibition in these cells. Given these data, resveratrol may sensitize colorectal cancer cells to cetuximab via upregulating connexin 43 to inhibit the Akt pathway.

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Hydroxyflutamide affects connexin 43 via the activation of PI3K/Akt-dependent pathway but has no effect on the crosstalk between PI3K/Akt and ERK1/2 pathways at the Raf-1 kinase level in primary rat Sertoli cells

Cited by 22 publications

References 68 publications

Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium

Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium

Notch signaling regulates nuclear androgen receptor AR and membrane androgen receptor ZIP9 in mouse Sertoli cells

Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

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