Protein expression of PKCZ (Protein Kinase C Zeta), Munc18c, and Syntaxin-4 in the insulin pathway in endometria of patients with polycystic ovary syndrome (PCOS)

Abstract

Background: Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic disorder commonly associated with insulin resistance (IR). Previous studies indicate about the expression of molecules involved in the insulin pathway in endometria of women with PCOS-IR. Therefore, the aim of the present study was to evaluate the effect of insulin and testosterone in the expression of these proteins in the endometria and immortal endometrial stromal cell line (T-HESCs).

Methods: We examined the protein levels of Munc18c, PKC zeta, phospho-PKC Zeta, and Syntaxin-4. Protein levels were assessed by Western Blot and/or immunohistochemistry in proliferative endometria (NPE = 6) and in PCOS endometria with insulin resistance (PCOSE-IR = 6). We also evaluated whether high concentrations of insulin (100 nM) and/or testosterone (100 nM), during a 24 h stimulatory period, affected the expression of these proteins in an immortal endometrial stromal cell line (T-HESCs). Once stimulated, proteins were extracted from cells and were assessed by Western Blot analysis. Immunocytochemistry was performed to detect AR in T-HESC cells.

Results: Western Blot data showed decreased expression (p < 0,05) of Munc18c and phospho-PKC Zeta in PCOS-IR endometria (PCOSE-IR) with respect to the control (NPE). In the in vitro study, Western Blot analysis showed decreased levels of Munc18c, PKC Zeta and phospho-PKC Zeta with the different hormonal treatments when compared to the control condition (no hormonal stimulation) (p < 0,05). The AR was present in the endometrial stromal cell line (T-HESC).

Conclusion: The conditions of hyperinsulinism and hyperandrogenism present in PCOS-IR patients modulate the expression and/or phosphorylation of the proteins involved in the insulin pathway at the endometrial level. These data extend to the T-HESCs cells results, where insulin and testosterone exert an effect on both the expression and phosphorylation of proteins present in the pathway.

Figure 1

Immunohistochemistry for proteins of the insulin signaling pathway. Representative image from immunohistochemical detection of Munc18c (A), PKCζ (B), phospho-PKCζ (C) and Syntaxin-4 (D) in paraffin wax sections of proliferative endometria (n = 6) (left panel) and PCOSE-IR (n = 6) (right panel). The hematoxylin/eosin staining showed comparable architecture in the two analyzed groups. Positive staining was detected in epithelial and stromal cells of all studied endometria for all antigens. Magnification 400× in all panels. Inserts show negative controls for immunohistochemical assays. Scale bars represent 50 μm.

Figure 2

Western blot analysis in endometria from proliferative phase (NPE) and from PCOS women with insulin resistance (PCOSE-IR). Equal amounts of protein were loaded in each lane. Munc18c (66 kDa), PKCζ (68 kDa), phospho-PKCζ (~68 kDa) and Syntaxin-4 (34 kDa) band intensities were quantified by scanning densitometry and normalized to intensities observed for β-actin as an internal control. A representative Western Blot for Munc18c, PKCζ, phospho-PKCζ and Syntaxin-4 is shown. The results are expressed as relative units (RU), and the values shown are mean ± SEM in Munc18c (n = 6), PKCζ (n = 6), phospho-PKCζ (n = 6) and Syntaxin-4 (n = 6). * p < 0.05 between Munc18c (NPE v/s PCOSE-IR) and phospho-PKCζ (NPE v/s PCOSE-IR).

Figure 3

Immunocytochemical detection of AR in T-HESCs cells.

Figure 4

The effects of insulin and/or testosterone on protein expression in T-HESCs cell cultures. Protein levels were assessed in cell exposed to 100 nM insulin (HI), 100 nM testosterone (HA), 100 nM insulin plus 100 nM testosterone (HI + HA), aand control cells. Representative Western Blot is shown below the bar graphs. Equal amounts of cell lysate were loaded in each lane, all the proteins were detected according to molecular mass: Munc18c (66 kDa) (A), PKCζ (68 kDa) (B), phospho-PKCζ (~68 kDa) (C) and Syntaxin-4 (34 kDa) (D). Band intensities were quantified by scanning densitometry and normalized to intensities observed for β-Actin. Results were expressed as relative units (RU), and the values are shown as mean ± SEM, * p < 0.05 between control and each hormonal treatment.