REPRODUCTIONRESEARCH

KCNH1 potassium channels are expressed in cervical cytologies from pregnant patients and are regulated by progesterone

A Ramı´rez, L M Hinojosa1, J d J Gonzales1, D Montante-Montes2, B Martı´nez-Benı´tez2, R Aguilar-Guadarrama2, A Gamboa-Domı´nguez2, F Morales3, A Carrillo-Garcı´a4, M Lizano4, R Garcı´a-Becerra5,LDı´az5,AYVa´zquez-Sa´nchez and J Camacho Department of Pharmacology, Centro de Investigacio´n y Estudios Avanzados del IPN, Avenida Instituto Polite´cnico Nacional 2508, Mexico City 07360, Mexico, 1Departamento de Ginecologı´a, Hospital General ‘Dr Manuel Gea Gonza´lez’, Calzada de Tlalpan 4800, Mexico City 14080, Mexico, 2Departamento de Patologı´a, Instituto Nacional de Ciencias Me´dicas y Nutricio´n ‘Salvador Zubira´n’, Vasco de Quiroga 15, Tlalpan, Mexico City 14000, Mexico, 3Departamento de Oncologı´a, Instituto Nacional de Cancerologı´a, Av. San Fernando No. 22, Col. Seccio´n XVI, Tlalpan, Mexico City 14080, Mexico, 4Unidad de Investigacio´n Biome´dica en Ca´ncer, Instituto Nacional de Cancerologı´a, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Av. San Fernando No. 22, Col. Seccio´n XVI, Tlalpan, Mexico City 14080, Mexico and 5Departamento de Biologı´a de la Reproduccio´n, Instituto Nacional de Ciencias Me´dicas y Nutricio´n ‘Salvador Zubira´n’, Vasco de Quiroga 15, Tlalpan, Mexico City 14000, Mexico Correspondence should be addressed to J Camacho; Email: [email protected]

Abstract

Potassium voltage-gated channel, subfamily H (eag-related), member 1 (KCNH1) potassium channels are potential tumour markers and cancer therapeutic targets and are up-regulated by oestrogens and human papilloma virus (HPV) oncogenes. However, the role of KCNH1 in normal tissues is poorly understood, and its expression in pregnancy is unknown. We wondered whether KCNH1 channels are

expressed in cervical cells from pregnant patients and whether progesterone (P4) regulates KCNH1. The association with HPV was also investigated. KCNH1 expression was studied by immunocytochemistry in liquid-based cervical cytologies; 93 samples were obtained from pregnant patients at different trimesters, and 15 samples were obtained from non-pregnant women (controls). The presence of HPV was studied by PCR with direct sequencing and nested multiplex PCR. HeLa cervical cancer cells were transfected with

human progesterone receptor-B (PR-B) and treated with P4. KCNH1 mRNA expression in these cultures was studied by real-time PCR. KCNH1 protein was detected in 100% of the pregnancy samples and in 26% of the controls. We found 18 pregnant patients infected with HPV and detected 14 types of HPV. There was no association between the percentage of cells expressing KCNH1 and either the presence

or type of HPV. P4 induced KCNH1 mRNA and protein expression in cells transfected with human PR-B. No regulation of KCNH1 by P4 was observed in non-transfected cells. We show for the first time the expression of an during human pregnancy at different

trimesters and KCNH1 regulation by P4 in human cells. These data raise a new research field for KCNH1 channels in human tissues. Reproduction (2013) 146 615–623

C Introduction a voltage-gated K channel that forms tetramers; each monomeric subunit has six putative transmembrane Ion channels play major roles in human physiology, domains, with an intracellular C- and N-terminus. In the including neural transmission, heart muscle contraction, N-terminal region, it has a calmodulin binding site and a insulin secretion and cell proliferation. Alterations in either the expression or the activityof ion channels are associated PAS domain (Per-Arnt-Sim), which has been suggested in with different diseases, including cardiac arrhythmias, other to be an oxygen sensor, and in the epilepsy, cystic fibrosis, hyperinsulinemia and deafness C-terminal region, it has several domains including one (Ashcroft 2006). Voltage-gated potassium channels have cyclic nucleotide binding domain, a nuclear localisation been associated with several diseases and represent targets signal and a calmodulin binding site (Scho¨nherr et al. for many disorders, including cancer (Wulff et al.2009). 2000, Bauer & Schwarz 2001, Downie et al. 2008, Wulff Ether a`-go-go-1 (EAG1, KCNH1, Kv10.1)is et al. 2009, Rodrı´guez-Rasgado et al. 2012).

q 2013 Society for Reproduction and Fertility DOI: 10.1530/REP-13-0318 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/30/2021 10:29:15PM via free access 616 A Ramı´rez and others

KCNH1 has gained great interest in oncology and its and are more commonly found in cervical cells from expression has also been proposed as a cervical patients taking oestrogens (Pardo et al. 1999, Dı´az dysplasia marker (Ortiz et al. 2011). In addition, these et al. 2009, Ortiz et al. 2011). Therefore, to gain channels are up-regulated by oestrogens and human insight into the potential role of KCNH1 in normal papilloma virus (HPV) oncogenes (Dı´az et al. 2009) and human physiology, we tested whether KCNH1 have been reported in almost 50% of patients taking channels are expressed in cervical cells under an oestrogens (Ortiz et al. 2011). Distribution of KCNH1 in oestrogen- and P4-enriched physiological environment normal human tissues is mainly restricted to the brain, such as human pregnancy. myoblasts and adrenal gland and to the reproduction- associated organs placenta and testes (Occhiodoro et al. 1998, Pardo et al. 1999, Hemmerlein et al. 2006, Dı´az Subjects and methods et al. 2009). Nevertheless, the precise role of KCNH1 in Biological samples normal tissues is nearly unknown. During pregnancy, the cervix undergoes marked Liquid-based cervical cytologies were obtained from proliferation (Burger & Sherwood 1995, Cunningham 93 Mexican-Mestizo pregnant patients (14–37 years old) et al. 2010), is under strong hormonal regulation and attending the General Hospital Dr Manuel Gea Gonza´lez in exhibits extensive remodelling, which includes soft- Mexico City, following local ethics considerations (protocol ening, ripening, dilation/labour and post partum repair number 11-29-2010, approved by the Institutional Review (Liggins 1978, Leppert 1995, Word et al. 2007, Timmons Board). Patients were in the first, second or third trimester of et al. 2010). The cervix is affected by increased cell pregnancy. Fifteen cervical cytologies from non-pregnant women (control patients with normal pap smears and no proliferation and decreased apoptosis in epithelial and cervical pathology) attending the Instituto Nacional de stromal cells; the peptide hormone relaxin, progesterone Cancerologı´a in Mexico City were also obtained, following (P4) and oestrogens contribute to the process by local ethics considerations (protocol number 010/024/ promoting cell proliferation and/or inhibiting apoptosis OMI/CB/622, approved by the Institutional Review Board). (Zhao et al. 2001, Lee & Sherwood 2005, Lee et al. All patients provided written informed consent, and none of 2005). These hormonal effects are most pronounced them had undergone any cervicovaginal treatment. Two during late pregnancy in rats, when the rate of cervical samples per patient were taken. One sample was obtained growth is higher (Zarrow & Yochim 1961, Zhao et al. using an endocervical brush and was used to study the 2001). The subtypes of oestrogen receptors ERa and ERb presence and type of HPV. The other sample was taken using are present in the human cervix and have been found to a broom-type cytobrush for liquid-based cervical cytology and undergo dynamic changes in expression. During term was used to study KCNH1 expression. pregnancy, the ERb mRNA expression levels are increased, while the ERa mRNA expression levels remain unchanged. However, after parturition, ERb and KCNH1 immunocytochemistry ERa expression decreases to the levels observed in Specific anti-EAG1 MABs were kindly provided by Walter non-pregnant women (Wang et al. 2001). Stu¨hmer and Luis Pardo (Max-Planck Institute for Experimental P4 is a very important steroid hormone in pregnancy Medicine, Go¨ttingen, Germany). This antibody has been because it promotes the morphological changes in the extensively validated. The antibody has also been tested for cervix and related tissues that help maintain pregnancy. specificity. It does not bind to Eag-related (Erg) channels P4 also inhibits smooth muscle contractions and the and discriminates between Eag1 and Eag2 (Go´mez-Varela et al. immune system, thus promoting an anti-inflammatory 2007). Using this antibody, we previously reported KCNH1 environment that helps maintain uterine quiescence protein expression in Chinese hamster ovary cells transfected (Hardy et al. 2006, Abeldazim et al. 2012, Byrns 2013). with KCNH1 (in contrast to no KCNH1 staining observed in non-transfected cells) and in human placenta and brain The predominant P4 receptor during human pregnancy is the B isoform (Goldman & Shalev 2007). primary cultures from cervical cancer biopsies, cervical tissues Some ion channels have been studied in the from cervical intraepithelial neoplasia, cervical cancer cell lines and human cervical cytologies (Farias et al. 2004, Dı´az myometrium during pregnancy (Suzuki & Takimoto et al. 2009, Ortiz et al. 2011). Normal keratinocytes (i.e. those 2005, Xu et al. 2011). 3, 4, 5 and 8 are not expressing KCNH1 mRNA) that were incubated with the involved in fluid homoeostasis in pregnant and peri- primary anti-EAG1 antibody did not exhibit KCNH1 protein partum cervices, thus facilitating water transport across expression (Ortiz et al. 2011). KCNH1 immunochemistry was the cervical epithelium (Anderson et al. 2006). However, performed as described previously (Dı´az et al. 2009, Ortiz et al. despite the importance of proliferation and apoptosis 2011). After standard treatments with anti-EAG1 antibody in cervical epithelial cells, no reports exist on the (1:100) and the mouse/rabbit secondary antibody (Biocare expression of ion channels that are associated with Medical, Concord, CA, USA), slides were washed followed by proliferation/apoptosis in cervical cells during human a 10-min incubation with peroxidase-linked polymer mouse/ pregnancy. KCNH1 channels have been associated with rabbit (Biocare Medical). Specific staining reactions were tumour cell proliferation, are regulated by oestrogens completed by incubating the slides in the presence of

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3,30-diaminobenzidine in reaction buffer, which was observed Transient human PR transfection as a brown staining. Sections were counterstained with The expression vector for human PR (pLEN-hPR ) used to haematoxylin (Dako, Glostrup, Denmark), washed with buffer B transfect HeLa cells was provided by Dr A J Cooney (Baylor and subsequently exposed to ethanol (Sigma–Aldrich), College of Medicine). Cells were transfected using PolyFect absoluteo alcohol (J T Baker, Phillipsburg, NJ, USA), xylene/ (Qiagen, Inc.), as described previously (Dı´az et al. 2009). absolute alcohol (1:1) and xylene (Sigma–Aldrich). Slides were The transfection of PR was confirmed using qRT-PCR. mounted with Entellan resine (Merck) and observed using a HeLa WT cells and PR-transfected cells were incubated Nikon Eclipse E50i microscope (Nikon, New York, NY, USA). for 24 h in the presence of increasing concentrations of P4 K K Brown immunostaining revealed KCNH1 expression. Slides (1!10 10–1!10 7 M) or vehicle (ethanol). were blindly analysed by two pathologists. Images were obtained using a digital COOLPIX P5100 Nikon camera. HeLa cervical cancer cells were purchased from American Real-time PCR Type Culture Collection (ATCC, Manassas, VA, USA) and After exposing the cells to the described treatments, medium transfected with progesterone receptor-B (PR-B), grown on was aspirated and RNA was extracted using TRIzol reagent. glass coverslips and cultured in the presence of either P4 cDNA was produced using 3 mg total RNA and the transcriptor ! K8 (1 10 M) or ethanol (vehicle) for 48 h. Cells were RT system (Roche Diagnostics). Real-time PCR was performed processed as described earlier for KCNH1 immunocytochem- using the LightCycler 2.0 from Roche, according to the istry. Quantification of immunostaining was performed using following protocol: activation of Taq DNA polymerase and digital images of systematic, randomly selected fields. Five to DNA denaturation at 95 8C for 10 min and 45 amplification seven fields of epithelial cells were measured using ImageJ cycles consisting of 10 s at 95 8C, 30 s at 60 8C and 1 s at 72 8C. Software (NIH, Bethesda, MD, USA). The signal intensity was The following primer sequences were used: PR, tca agc ttc aag measured as pixels per cell nuclei area. tta gcc aag a, gac ttc gta gcc ctt cca aa; KCNH1, cct gga ggt gat cca aga tg, cca aac acg tct cct ttt cc. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was HPV detection and typing used as an internal control: agc cac atc gct gag aca c, gcc Cervical samples were digested with 1 ml lysis buffer caa tac gac caa atc c. (10 mmol/l Tris–HCl, pH 8.0, and 0.1 mol/l EDTA (Gibco-BRL), pH 8.0, 0.5% SDS, 200 mg/ml proteinase K and 20 mg/ml RNase A) at 55 8C for 3 h. DNA was extracted with Statistical analysis phenol/chloroform and precipitated with ethanol as described Statistical analysis was performed with the c2 test, Student’s by Sambrook et al. (1989). To test the DNA suitability for PCR t-test and one-way ANOVA using the SPSS statistical program. amplification, the DNA obtained was amplified for b-globin A P value !0.05 was considered significant. (PCO4/GH2O) under conditions described by Resnick et al. (1990). Samples were later submitted for HPV amplification using two sets of the following universal primers recognising Results distinct size fragments of the L1 gene: L1C1/L1C2.1/L1C2.2 KCNH1 expression in cervical cytologies from and MY09/MY11 (van den Brule et al. 1990, Snijders et al. pregnant patients 1990, Yoshikawa et al. 1991). Amplification using MY09 and MY11 produced a conserved 450 bp fragment from the L1 We first examined the expression of KCNH1 channels in gene, and amplification using the LC primers produced a cervical cytologies from pregnant women at different 250 bp fragment of the L1 gene located upstream of the MY trimesters (Fig. 1). KCNH1 expression was observed in sequence. The following PCR protocol was used: 94 8C for intermediate cells and superficial cells; however, the cells that were negative for KCNH1 were most 10 min, 38 cycles at a Tm of either 55 8C (for MY primers) or 49 8C (for LC primers) for 50 s and 72 8C for 7 min. DNA commonly observed in superficial cells of the cervical extracted from CaSki and Hela HPV-containing cells (from layer. Previously obtained placental sections (Ortiz et al. ATCC) were used as controls. Mixtures without DNA were 2011) were used as a positive control in which the included as negative controls. HPV PCR products were expected KCNH1 expression was observed, whereas no electrophoresed on a 1.2% agarose gel (Gibco-BRL) and staining emerged in the absence of the primary antibody visualised with ethidium bromide staining. HPV typing was (Fig. 1). We observed KCNH1 expression in all the carried out using direct sequencing of PCR products with the samples from pregnant patients (Table 1). KCNH1 BigDye Terminator v3.1 Cycle Sequencing Kit (Applied expression in normal cervical cytologies was reported Biosystems). The resulting sequences were analysed using the in 27% of the patients (Ortiz et al. 2011). Accordingly, BLAST NCBI GenBank program (http://www.ncbi.nlm.nih. we found that 26.6% of the samples from non-pregnant gov). Reagents were obtained from Sigma–Aldrich, unless women (i.e. control patients with pap smears that were otherwise indicated. Multiple HPV infections were analysed negative for intraepithelial lesions) were positive for through nested multiplex PCR described by Sotlar et al. (2004), KCNH1 (Table 1). based on consensus and type-specific primers, amplifying a Although all the samples from pregnant patients were region of E6/E7 . positive for KCNH1, the percentage of positive cells from www.reproduction-online.org Reproduction (2013) 146 615–623

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Trimester 1st 2nd 3rd

Figure 1 Detection of KCNH1 in cervical cytologies from pregnant patients. Individual examples are shown for the different trimesters of pregnancy: first tri- mester (2–12 weeks, nZ13), second trime- ster (13–24 weeks, nZ33) and third trimester (25–40 weeks, nZ47). KCNH1 immunostaining was observed in the cytoplasm, nucleus and membrane, as previously reported for several cell types (Dı´az et al. 2009, Ortiz et al. 2011). No staining was observed in cervical cells in Positive the absence of the primary antibody Negative control (lower panel, left). Normal human placenta control (placenta) was used as a positive control for KCNH1 expression and displayed clear immunostaining (lower panel, right), as previously reported (Dı´az et al. 2009, Ortiz et al. 2011). Magnification: upper panels, 200!; middle and lower panels, 400!. each sample ranged from 5 to 90%. This finding was expressing KCNH1 might be associated with the type consistent among samples from different trimesters of HPV. We did not identify any correlation between the (Fig. 2). Most of the cervical cytologies from non- percentage of KCNH1-positive cells and either the pregnant women were negative for KCNH1 expression, presence or type of HPV (Fig. 3). and none of them contained more than 66% of positive cells. By contrast, most of the samples from pregnant patients in the second and third trimesters displayed P4 regulates KCNH1 expression more than 67% of positive cells, with many samples The lack of association between KCNH1 expression and exhibiting 100% of KCNH1-expressing cells (Fig. 2). the presence of HPV prompted us to study KCNH1 Because HPV oncogenes regulate KCNH1 channel regulation by a very important hormone in human expression, we studied the potential presence and, in pregnancy, P4. We previously demonstrated that oestro- the corresponding cases, the type of HPV in cervical gens up-regulate KCNH1 mRNA expression in cervical cytologies from pregnant patients. cancer cells and that ERa is necessary for this regulation. We analysed KCNH1 mRNA and protein expression in the presence of P in HeLa cervical cancer cells using HPV detection in cervical cytologies from 4 pregnant patients real-time PCR and immunocytochemistry respectively. We used WT HeLa cells and HeLa cells over-expressing To determine whether KCNH1 expression might be due human PR-B. We did not observe the P4-mediated to HPV infection, we studied the presence and type regulation of KCNH1 expression in WT, non-transfected (i.e. low or high risk) of HPV in the cervical cytologies cells. By contrast, P4 induced a significant dose– from pregnant patients. We identified HPV in 19% response increase in KCNH1 mRNA expression in cells (18 of 93) of the samples, in accordance with previous transfected with human PR (Fig. 4A). We next tested reports (Hagensee et al. 1999, Medeiros et al. 2005, Castellsague´et al. 2009). The HPV type was defined by Table 1 KCNH1 in cervical cytologies from pregnant and direct sequencing of the PCR product and using the non-pregnant patients. nested multiplex PCR method. We identified 14 types of HPV distributed in 18 HPV-positive samples, with eight KCNH1-positive samples (%) samples displaying more than one HPV type (Fig. 3). We Pregnant 93/93 (100) next determined whether the percentage of cells Non-pregnant 4/15 (26.6)

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25 with oestradiol (Dı´az et al. 2009). However, the role No KCNH1 expression of KCNH1 channels in human placenta and testes KCNH1-positive cells per sample remains unknown. 20 5–33% One of the most striking features of KCNH1 channels 34–66% is its oncogenic potential and association with cancer 67–100% (Pardo et al. 1999, Farias et al. 2004, Hemmerlein et al. 15 2006, Ousingsawat et al. 2007, Dı´az et al. 2009, Asher et al. 2010). KCNH1 has been suggested to function as a tumour marker for different types of malignancies, 10 including cervical cancer (Pardo et al. 1999, Farias et al. Number of samples 2004, Hemmerlein et al. 2006). The expression of 5 KCNH1 in normal cervical cytologies (negative pap smears) has been observed in 27% of the patients, whereas it is found in 67% of patients with low-grade 0 cervical intraepithelial lesions and in 92% of patients 1st 2nd 3rd with high-grade cervical intraepithelial lesions (Ortiz Non-pregnant Pregnancy trimester et al. 2011). In addition, KCNH1 has been reported in Figure 2 Pregnancy cervical cytologies exhibit a high percentage of almost 50% of patients taking oestrogens (Ortiz et al. KCNH1-positive cells. The percentage of KCNH1-positive cells was 2011). The oestrogenic regulation of KCNH1 channels counted, and each sample was grouped into one of the following has also been demonstrated in HeLa cervical cancer four levels of KCNH1 expression: No KCNH1 expression (0–4%), 5–33, 34–66, and 67–100% of positive cells. A 7

6 Low risk whether P4 also up-regulated channel protein expression High risk in PR-B-transfected cells. KCNH1 protein expression 5 was also increased in transfected cells upon P4 treatment (Fig. 4B, C, D, E and F). 4

3

Discussion 2 Here, we show the expression of an ion channel during 1 Number of HPV-positive samples human pregnancy at different trimesters. As previously 0 reported, we found KCNH1 expression in 26% of non- 42 43 44 62 67 72 81 16 18 31 51 56 66 6/11 pregnant patients (Ortiz et al. 2011). By contrast, Type of HPV KCNH1 expression was observed in 100% of the cervical samples from pregnant patients. The cervix B undergoes marked proliferation during pregnancy and is 90 under strong hormonal regulation (Burger & Sherwood 80 1995, Cunningham et al. 2010). KCNH1 channels have 70 been associated with cell proliferation and are up-regu- 60 lated by oestrogens (Pardo et al. 1999, Farias et al. 2004, 50 Hemmerlein et al. 2006, Ousingsawat et al. 2007, Dı´az 40 et al. 2009, Asher et al. 2010). Thus, the expression 30 of KCNH1 in all the cervical samples from pregnant 20 patients strongly suggests a role for this channel during 15 human pregnancy. Cells expressing KCNH1 (%) 10 The distribution of the KCNH1 channel in normal 5 91 tissues is restricted and is mainly expressed in the brain (Pardo et al. 1999, Hemmerlein et al. 2006). Interest- HPV- Low High ingly, KCNH1 is also expressed in tissues/organs negative risk-HPV risk-HPV associated with reproduction, such as the placenta and testes (Pardo et al. 1999, Hemmerlein et al. 2006, Dı´az Figure 3 KCNH1 expression is not associated with HPV infection in et al. 2009). Oestrogens up-regulate KCNH1 mRNA cervical samples from pregnant patients. We found HPV infection in 18 patients, and we found 14 different HPV types (A). Co-infections with expression in placental trophoblast primary cultures more than one HPV type were observed in eight patients. The percentage and elicit outward potassium currents that resemble of cells expressing KCNH1 in each sample was not associated with either KCNH1 channel activity in trophoblasts incubated the presence or type of HPV detected (B) (PZ0.715). www.reproduction-online.org Reproduction (2013) 146 615–623

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cells. Such regulation is strongly associated with the presence of ERa (Dı´az et al. 2009). Thus, KCNH1 channels have also been proposed as cervical dysplasia markers and potential risk indicators of the disease. Nevertheless, the role of KCNH1 channels in normal A 2.5 Wild-type cells * Transfected cells human cervices is elusive. 2.0 * Cervical epithelial proliferation decreases while apoptosis increases in post partum rats, with a con- 1.5 comitant decrease in ERa after delivery. The KCNH1 expression observed in 100% of the samples from 1.0 pregnant patients might be associated with epithelial proliferation, hormone levels and the presence of ERa 0.5 and PR-B. KCNH1 functions to maintain the homo-

Relative KCNH1 expression eostasis of oxygen in tumours by increasing VEGF and HIF-1 expression (Downie et al. 2008). During preg- C –10 –9 –8 –7 nancy, the cervix undergoes vascular changes to increase irrigation of the tissue; thus, KCNH1 might have a role P4 (log/molar) in the regulation of cervical vascular homoeostasis Vehicle-treated cells P4-treated cells during pregnancy. B C It is worth mentioning that the role of the KCNH1- mediated current in normal tissues is poorly understood. KCNH1 channel activity has been proposed to provide the hyperpolarising current required in myoblasts just before cell fusion (Occhiodoro et al. 1998). A nuclear localisation sequence is present in KCNH1 and pot- assium currents resembling Kcnh1 channel activity blocked by astemizole have been reported in the nuclear D E membrane (Chen et al. 2011). However, the potential role of KCNH1 in the nucleus remains elusive. In normal tissues, KCNH1 is mainly expressed in the brain (Pardo et al. 1999, Hemmerlein et al. 2006). Interestingly, Kcnh1 knockout mice have been produced, but the animals did not show any major abnormality at the CNS level (Ufartes et al. 2013). It is also known that ion channels have several non-conducting functions includ- F 40 * ing interactions with other proteins like enzymes or cytoskeleton proteins (Kaczmarek 2006). Actually, non- 30 conducting KCNH1 potassium channels are still able to induce tumour formation (Downie et al. 2008). 20 Definitely, it would be very interesting to study Kcnh1

(pixel/area) expression during pregnancy in the Kcnh1 knockout 10 Staining intensity model to gain insights into the potential role of KCNH1 channels in the cervix during pregnancy.

Medium Vehicle P4 Because KCNH1 expression is up-regulated by HPV oncogenes (Dı´az et al. 2009), we tested whether this Figure 4 KCNH1 expression is regulated by progesterone. (A) KCNH1 variation might be due in part to HPV infection. We did mRNA expression is significantly increased after P4 treatment in PR-B- not observe an association between KCNH1 expression transfected cells. Relative KCNH1 mRNA levels were obtained by normalising against GAPDH mRNA expression. Vehicle values were and either the presence or type of HPV. In the case of set to 1. Data are expressed as the meanGS.D. of triplicates from the non-pregnant women, probably it was only an early HPV same experiment. The graph is representative of two independent infection that did not affect Kcnh1 expression, as it is experiments. *P!0.05 vs control. (B, C, D, E and F) KCNH1 protein known that persistent or recurrent HPV infections are expression (brown immunostaining) is enhanced by P4. HeLa cells needed to modify the cellular phenotype. It will be transfected with human PR-B were either treated with vehicle (B and D) K important to study Kcnh1 expression in cervical pap or incubated in the presence of progesterone (1!10 8 M) for 48 h (C and E). Densitometric analysis showed a significant increase in KCNH1 smears from patients who have been infected several protein expression in cells treated with progesterone (F). *P!0.05 vs times with HPV. On the other hand, because all the vehicle and medium. Approximately 120 cells were analysed in each pregnant patients were positive for Kcnh1, perhaps group. Magnification: (B and C), 200!; (D and E), 400!. KCNH1 regulation by hormones is stronger than the

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Downloaded from Bioscientifica.com at 09/30/2021 10:29:15PM via free access KCNH1 channels in human pregnancy 621 regulation by HPV. Actually, here, we observed a clear cervical softening is associated with decreased collagen increase in KCNH1 expression in cervical cells treated organisation, which is characterised by increased with P4, and Kcnh1 expression has been reported to be collagen solubility and decreased collagen concen- regulated by oestrogens (Dı´az et al. 2009). These results tration. In pregnancy, collagen solubility increases suggest that KCNH1 expression in cervical cells of early. In the third trimester, up to 90% of cervical pregnant women is associated with hormonal regulation collagen is soluble; thus, its concentration decreases rather than HPV infection. w50% compared with non-pregnant women (House We also found that the number of cells that express et al. 2009, Myers et al. 2009). Human KCNH1- KCNH1 varies from 5 to 90% in pregnant women. While mediated potassium currents are decreased in cells most of the cervical cytologies from non-pregnant grown on collagen (Toral et al. 2007). Thus, a plausible women were negative for KCNH1 expression and none scenario is that the up-regulating effects of P4 on KCNH1 of them showed more than 66% of positive cells, most of mRNA and protein expression are related to the the pregnancy samples from the second and third inhibitory effect of P4 on collagen organisation/ trimesters displayed more than 67% of KCNH1-positive concentration. cells with many samples exhibiting 100% of KCNH1- There are several PR isoforms including PR-A positive cells. We did not find significant differences (N-terminal truncated receptor), PR-B (full-length between the percentage ranges of positive cells during receptor) and PR-C (Evans 1988, Goldman & Shalev different trimesters (Fig. 2). Nevertheless, this lack of 2007). PR-B is the principal mediator of P4 actions and is significant difference might be due to either the number the predominant form during pregnancy (Graham & of samples in each trimester or the precise pregnancy Clarke 2002, Goldman & Shalev 2007). PR regulates time. KCNH1 expression in patients at the end of one genes that encode various ion pumps and channels trimester might be very similar to channel expression (Connaghan et al. 2010). For example, P4 was shown to in the early stages of the following trimester. The modulate the expression of TRPV4 channels via the PR pregnancy time of some patients was very close to the (Jung et al. 2009). Taken together, these results suggest end of the trimester, and it was at the beginning of the that P4 also regulates KCNH1 expression via PR-B, as second or third trimester in other cases. Thus, it will be shown in this study. very important to study KCNH1 expression during Rat KCNH1-mediated potassium currents are inhib- pregnancy in a more detailed manner. For example, ited in Xenopus laevis oocytes after incubation with P4 dividing the patients in groups of 12, 24 or 36 weeks of (Bru¨ggemann et al. 1997). Nevertheless, in X. laevis pregnancy or using days of gestation instead of trimesters oocytes, P4 binds to a membrane PR, which is the only may give more insight into the regulation of KCNH1 PR type identified in Xenopus (Sadler & Maller 1982). expression during pregnancy. In addition, more Here, we studied human KCNH1 channels and quantitative studies would be useful. For example, human PR-B, but more studies are needed to elucidate quantitative analyses could be performed to evaluate the mechanism of the P4-mediated regulation of KCNH1 mRNA or protein expression using real-time KCNH1 expression. PCR or western blot techniques respectively. Doing so Our results suggest a novel role for KCNH1 channels may lead to identification of a closer relationship in cervical physiology during pregnancy, which is between channel expression and gestation time. most likely associated with hormonal regulation and Hormone blood levels could also be quantified to assess raises a new research field for KCNH1 channels in a potential association with KCNH1 expression. healthy human tissues. To our knowledge, this study is the first to show the regulation of KCNH1 mRNA and protein expression by Declaration of interest P4. We did not observe hormonal regulation in HeLa WT cells. By contrast, P4 increased KCNH1 expression in The authors declare that there is no conflict of interest that cells transfected with human PR-B, strongly suggesting could be perceived as prejudicing the impartiality of the that this receptor is necessary for such regulation. P4 is research reported. essential in pregnancy, and high levels of this hormone are maintained during gestation (Hardy et al. 2006). Funding Cervical remodelling during pregnancy depends on P4 because it can modulate the expression of some This work was partially supported by the Consejo Nacional de components of the extracellular matrix (ECM), thus Ciencia y Tecnologı´a (Conacyt, grant number 141126) to inhibiting hyaluronate expression (Tanaka et al. 1994, J Camacho. 1997) and increasing collagenase, elastase and metallo- proteinase levels (Andersson et al. 2008). Because the cervical tissue mechanical properties are related to Acknowledgements the ECM (Myers et al. 2008) and because the ECM The authors thank Walter Stu¨hmer and Luis Pardo (Max-Planck is the major component of cervical tissue (Leppert 1995), Institute fu¨r Experimentelle Medizin, Go¨ttingen, Germany) www.reproduction-online.org Reproduction (2013) 146 615–623

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