Endometriosis and Type I Interferon & Characterization of a Mammalian Flippase

Research output: Book/anthology/dissertation/reportPh.D. thesis

Documents

  • Anna Lindeløv Vestergaard, Denmark
  • Department of Molecular Biology
  • Department of Physiology and Biophysics
  • Aarhus Graduate School of Science/AGSoS
  • Aarhus University

Endometriosis and Type I Interferon

 

Endometriosis is a painful chronic disease in which endometrium-like lesions are located ectopically, frequently in the pelvic cavity but also in more distant sites. The pathogenesis of endometriosis is unclear and involves complex hormonal, genetic, environmental, malignant-like, and immunological mechanisms. Evidence points towards a possible involvement of the type I interferons (IFNs).

The expression of 84 genes related to the IFN-system were profiled using a specific qRT-PCR array on tissue samples of endometrium of healthy control women (C) and in eutopic endometrium (Eu) and ectopic endometriosis lesions (Ec) of women with endometriosis. The four genes BST2, COL16A1, ISG20 and HOXB2 appeared significantly differentially regulated between the groups. However, after a thorough investigation of appropriate reference genes for normalization, validation by qRT-PCR confirmed only that ISG20 and HOXB2 were significantly downregulated in the Ec group compared with the Eu and C groups. BST2 and COL16A1, as well as the highly IFN-stimulated genes ISG12A and 6-16, displayed merely insignificant variation between the groups.

Endometriosis displays malignant-like features such as invasiveness and metastasis, and malignant features of well-known proto-oncogenes and tumor suppressor genes in endometriosis lesions were therefore studied. Purified DNA from lesions displayed no promoter hypermethylation of the genes APC, CDKN2A, PYCARD, RARB, RASSF1, and ERa, when analyzed by bisulfate PCR and melting curve analysis. Also, no mutations of BRAF, HRAS, NRAS, CTNNB1, CDK4, FGFR3, PIK3CA, P53, and PTEN were detected by PCR denaturing gradient gel electrophoresis analysis. A well-known cancer-associated mutation in KRAS was detected in a single endometriosis lesion.

Human papillomavirus (HPV) is the causative agent of cervix cancer, and DNA viruses might play a role in endometriosis. DNA purified from tissue samples were subjected to highly sensitive PCR tests detecting HPV types, the herpes family viruses HSV-1 and -2, CMV, and EBV, and the polyomaviruses SV40, JCV, BKV, KIV, WUV, and MCV. The prevalence of pathogenic DNA viruses in C and Eu groups was generally low (0–10%), and no viruses were identified in endometriotic lesions.

The results obtained in this PhD project do not point towards a direct involvement of type I IFNs or DNA viruses in endometriosis, and the possible contribution by a number of malignant features was eliminated. The established downregulation of ISG20 and HOXB2 transcription and the putative existence of other well-known malignant features should be studied further in the investigation of pathogenic mechanisms of endometriosis.

 

Characterization of a Mammalian Flippase

 

P-type ATPases transport specific substances across biological membranes at the cost of ATP hydrolysis. They are directly involved in essential physiological functions and are also implicated in a number of pathological conditions, such as Menkes, Wilson, and Byler disease, migraine, and parkinsonism. The P4-type ATPases, also named the flippases, translocate aminophospholipids rather than ions to the cytosolic side of the membrane bilayer and are allegedly crucial for the essential asymmetry of bilayered biological membranes. Members of the CDC50 protein family are believed to be chaperones or additional subunits to these enzymes. The molecular mechanism of the flippases is currently unknown, and whereas studies of the yeast family members have provided some functional knowledge on the matter, the mammalian homologs are still fairly uncharacterized.

The murine flippase Atp8a1 was cloned and expressed as a histidine-tagged recombinant protein in Sf9 insect cells in the baculovirus expression system. The murine Cdc50a and -b proteins were also cloned and expressed as hemagglutinin-tagged recombinant proteins. Cdc50a and -b co-purified with Atp8a1 on a nickel resin, demonstrating a strong association between these proteins. Atp8a1 could be phosphorylated by [g32P]-ATP in the presence of Mg2+. This phosphorylation was sensitive to hydroxylamine indicating the formation of the characteristic P-type ATPase acyl-phosphate intermediate. An Atp8a1 mutant with a substitution of the active site aspartate residue for an asparagine was phosphorylated to a lesser degree and was less sensitive to hydroxylamine. The co-expression of Cdc50a or -b had no apparent effect on the degree of phosphorylation of neither wild-type nor mutant Atp8a1.

Future studies characterizing the ATPase activity of Atp8a1 and the potential influence of the Cdc50 proteins should be conducted. The phospholipid substrate selectivity and the possible transport of counter-substrates should also be addressed. These studies will increase the knowledge on the biological functions of the flippases and create an important basis for investigating their structure and function in order to better understand their roles in physiological and pathological mechanisms.

Original languageEnglish
Place of publicationAarhus Universitet
Number of pages159
Publication statusPublished - 2010

    Research areas

  • Endometrium, Endometriosis, Interferon, Virus, Flippase, Mammalian, ATPase, P-type, Baculovirus, PCR, Housekeeping genes, Isg12

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