Lysyl oxidase interacts with AGE signalling to modulate collagen synthesis in polycystic ovarian tissue - PubMed (original) (raw)

Lysyl oxidase interacts with AGE signalling to modulate collagen synthesis in polycystic ovarian tissue

Katerina K Papachroni et al. J Cell Mol Med. 2010 Oct.

Abstract

Connective tissue components--collagen types I, III and IV--surrounding the ovarian follicles undergo drastic changes during ovulation. Abnormal collagen synthesis and increased volume and density of ovarian stroma characterize the polycystic ovary syndrome (PCOS). During the ovulatory process, collagen synthesis is regulated by prolyl hydroxylase and lysyl oxidase (LOX) activity in ovarian follicles. LOX catalyzes collagen and elastin cross-linking and plays essential role in coordinating the control of ovarian extracellular matrix (ECM) during follicular development. We have recently shown accumulation of advanced glycation end products (AGEs), molecules that stimulate ECM production and abnormal collagen cross-linking, in ovarian tissue. However, the possible link between LOX and AGEs-induced signalling in collagen production and stroma formation in ovarian tissue from PCOS remains elusive. The present study investigates the hypothesis of AGE signalling pathway interaction with LOX gene activity in polycystic ovarian (PCO) tissue. We show an increased distribution and co-localization of LOX, collagen type IV and AGE molecules in the PCO tissue compared to control, as well as augmented expression of AGE signalling mediators/effectors, phospho(p)-ERK, phospho(p)-c-Jun and nuclear factor κB (NF-κB) in pathological tissue. Moreover, we demonstrate binding of AGE-induced transcription factors, NF-κB and activator protein-1 (AP-1) on LOX promoter, indicating a possible involvement of AGEs in LOX gene regulation, which may account for the documented increase in LOX mRNA and protein levels compared to control. These findings suggest that deposition of excess collagen in PCO tissue that induces cystogenesis may, in part, be due to AGE-mediated stimulation of LOX activity.

© 2009 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

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Figures

Fig 1

Fig 1

Immunodetection of collagen type IV and LOX in PCO and control samples. Immunohistochemistry was performed on sections from stromal (top panels) or follicular (bottom panel) ovarian tissue, which were stained for collagen type IV (A) and LOX (B). Increased selective staining is observed in PCO sections (magnification ×100). Pictures are representative of 20 sections. (C) Immunohistochemical detection of AGEs in polycystic ovaries [24]. Positive staining is observed in the follicular cell layers (granulosa and theca layers), luteinized cells and in endothelial cells of polycystic ovaries, with stronger staining intensity observed in granulosa cells compared to normal tissue (_P_= 0.036). AGE immunoreactivity positively correlates with LOX staining in granulosa cells (_R_= 0.285, _P_= 0.041). (D) Western immunoblotting of 40 μg of whole-cell extract from control and PCO tissue against anti-collagen type IV and anti-LOX antibodies. Collagen type IV protein expression is increased by 3-fold and LOX protein expression by approximately 4-fold in the PCO tissue relative to control.

Fig 3

Fig 3

Induced NF-κB and AP-1 binding to LOX promoter in PCO tissue. (A) Line diagram of part of the LOX promoter. The binding sites for the NF-κB and AP-1 transcription factors are indicated, as well as the restriction enzymes are used to isolate the respective DNA fragments from E. coli transfected cells. (B, C) EMSA with the NF-κβ p65 DNA biotin-labelled probe (Bi) or the AP-1/c-Jun biotin-labelled probe (Ci) and whole-cell ovarian protein extract from human samples of healthy (control) and PCO tissue. Increasing concentrations (10, 20, 40 μg) of protein were used (lanes 1–3 for control and 4–6 for PCO tissue protein extract) and the LOX promoter – NF-κB and LOX promoter – AP-1 complex is indicated in the two panels, respectively. (Bii, Cii) Binding competition assays with 100-fold (lane 9) and 200-fold (lane 10) molar excess of specific unlabelled probe reacting with protein extract from PCO tissue. Note that in the lower panel (AP-1 probe, Cii), lane 9* contains 200-fold molar excess and lane 10* contains 100-fold molar excess of specific unlabelled probe. Lanes 11 and 12 correspond to reactions in which pre-incubation of control and PCO tissue protein extract with anti-NF-κB p65 or anti-p-c-Jun antibody was performed, before the addition of the labelled probe.

Fig 2

Fig 2

Immunodetection of p-ERK, p-c-Jun and NF-κB p65 in PCO and control samples. (A) Sections of ovarian tissue were immunostained for p-ERK, p-c-Jun and NF-κB p65. Increased staining is observed on the pathological tissue. Pictures are representative of 20 sections (magnification ×100). (B) Western immunoblotting of 40 μg of whole-cell extract from control and PCO tissue incubated with anti-p-ERK, anti-p-c-Jun and anti-NF-κB p65 antibodies. Protein levels of p-ERK are increased by 3-fold, of p-c-Jun 1.5-fold and of NF-κB p65 2-fold relative to control.

Fig 4

Fig 4

Induction of the LOX mRNA and LOX protein levels in the pathological ovarian tissue. (A) Real-time PCR was employed to achieve relative quantification of LOX mRNA in control and PCO tissue. Using the Pfaffl equation increase in LOX mRNA expression was calculated as 3-fold relative to control. Representative graph of the melting curves of the LOX and GAPDH PCR products. (B) LOX protein levels are increased by almost 4-fold in PCO tissue as revealed by Western immunoblotting.

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