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Protein kinases and phosphatases regulate cellular processes by reversible phosphorylation and dephosphorylation events. CPPED1 is a recently identified serine/threonine protein phosphatase that dephosphorylates AKT1 of the PI3K-AKT signalling pathway. We previously showed that CPPED1 levels are down-regulated in the human placenta during spontaneous term birth. In this study, based on sequence comparisons, we propose that CPPED1 is a member of the class III phosphodiesterase (PDE) subfamily within the calcineurin-like metallophosphoesterase (MPE) superfamily rather than a member of the phosphoprotein phosphatase (PPP) or metal-dependent protein phosphatase (PPM) protein families. We used a human proteome microarray to identify 36 proteins that putatively interact with CPPED1. Of these, GRB2, PAK4 and PIK3R2 are known to regulate the PI3K-AKT pathway. We further confirmed CPPED1 interactions with PAK4 and PIK3R2 by coimmunoprecipitation analyses. We characterized the effect of CPPED1 on phosphorylation of PAK4 and PIK3R2 in vitro by mass spectrometry. CPPED1 dephosphorylated specific serine residues in PAK4, while phosphorylation levels in PIK3R2 remained unchanged. Our findings indicate that CPPED1 may regulate PI3K-AKT pathway activity at multiple levels. Higher CPPED1 levels may inhibit PI3K-AKT pathway maintaining pregnancy. Consequences of decreased CPPED1 expression during labour remain to be elucidated.

Human parturition is a complicated biological process and the molecular mechanisms regulating the onset of labor are not well understood. Several studies propose that gene variants, proteins and miRNAs play a major role in maintaining pregnancy and aberrations in protein and miRNA levels may lead to pregnancy complications. The levels of proteins and miRNAs could prove to be useful biomarkers for monitoring the onset of labor. In this study, using proteomics and a genome analysis, we identified Calcineurin-like phosphoesterase domain-containing protein (CPPED1), as a potential biomarker of labor. CPPED1 was found to be downregulated in spontaneous term placentas compared with placentas from elective deliveries. The siRNA knockdown of CPPED1 in trophoblasts derived cells mostly affects pathways related to inflammation and angiogenesis, and these functions may be involved in the induction of labor or in maintaining pregnancy. In bladder cancer cells, CPPED1 has been shown to dephosphorylate AKT1 on the Ser-473 residue and to promote apoptosis via the AKT/PI3K pathway. In our studies using cells of placental origin AKT1 phosphorylation levels were unaffected by CPPED1 expression, whereas its silencing led to the upregulation of negative regulators of the PI3K pathway. In a comparative miRNAomics screen of spontaneous term vs. elective term placentas, we identified 54 differentially expressed miRNAs, out of which 23 miRNAs were upregulated and 31 were downregulated by at least 1.5-fold. Among the upregulated miRNAs, miRNA-371a-5p targets CPPED1 and post-transcriptionally regulates its expression by binding to its 3’UTR region. In addition, we found that CPPED1 and miR-371a-5p levels inversely correlate with each other in spontaneous and elective term placentas. Measuring the levels of CPPED1 and miR-371a-5p levels in maternal blood could be a potential diagnostic biomarker for labor. In order to elucidate the functions of CPPED1 we used a protein microarray to, Tiivistelmä Ihmisen synnytyksen käynnistyminen ja synnytys ovat monimutkaisia biologisia prosesseja, joiden kaikkia taustalla olevia molekyylimekanismeja ei vielä tunneta. Useat tutkimukset osoittavat, että tietyillä proteiineilla ja mikroRNA-molekyyleillä on tärkeä rooli raskauden ylläpitämisessä. Näiden proteiini- ja mikroRNA-määrien poikkeamat voivat johtaa raskauskomplikaatioihin. Proteiinien ja mikroRNA:n määrien mittaamista voitaisiin siten käyttää mahdollisina raskauskomplikaatioiden ja synnytyksen biomarkkereina. Tässä tutkimuksessa löysimme proteomiikan ja genomin laajuisen assosiaatiotutkimuksen (GWAS) avulla entsyymin nimeltä Calcineurin-like phosphoesterase domain-containing protein 1 (CPPED1), joka mahdollisesti voi toimia spontaanin synnytyksen biomarkkerina. CPPED1-tasot laskivat spontaaneissa synnytyksissä, kun niistä saatuja istukoita verrattiin keisarinleikkauksen kautta saatuihin istukkoihin. Kun CPPED1-geenin ilmeneminen hiljennettiin istukan trofoblastisoluissa, tulehduksiin ja verisuonten uudismuodostukseen liittyvät signalointireitit muuttuivat eniten. Näillä muutoksilla saattaa olla merkitystä raskauden kestoon. Aikaisemmassa tutkimuksessa on osoitettu, että virtsarakon syöpäsoluissa CPPED1 poistaa fosfaattiryhmän AKT1-entsyymin seriini-473 aminohapolta. Tämän fosfaatin poistaminen johti ohjelmoituun solukuolemaan AKT/PI3K-signalointireitin kautta. Meidän omissa tutkimuksissamme AKT1-entsyymin fosforylointi ei muuttunut istukan soluissa vaikkakin CPPED1:n määrä muuttui. Sen sijaan, CPPED1-määrän laskiessa PI3K-signalointireittiä negatiivisesti säätelevien proteiinien määrät lisääntyivät istukasta peräisin olevassa solulinjassa. Vertailevassa mikroRNAomiikassa, jossa vertailtiin alatiesynnytyksistä ja keisarinleikkauksista peräisin olevien istukoiden mikroRNA-molekyylejä, havaitsimme merkittäviä muutoksia 54 mikroRNA-molekyylien määrissä. Näistä 54 mikroRNA:stä 23 mikroRNA:n määrät nousivat ja 31 mikroRNA:n määrät laskivat spontaaneiden täysia

MicroRNAs (miRNAs) are important regulators of gene expression, and their expression is associated with many physiological conditions. Here, we investigated potential associations between expression levels of miRNAs in human placenta and the onset of spontaneous term birth. Using RNA sequencing, we identified 54 miRNAs differentially expressed during spontaneous term labor compared to elective term births. Expression levels of 23 miRNAs were upregulated, whereas 31 were downregulated at least 1.5-fold. The upregulated miRNA miR-371a-5p putatively targets CPPED1, expression of which decreases during spontaneous birth. We used a luciferase reporter–based assay to test whether a miR-371a-5p mimic affected translation when it bound to the 3′ untranslated region of CPPED1. In this setting, the miR-371a-5p mimic resulted in lower luciferase activity, which suggests that miR-371a-5p regulates levels of CPPED1. In conclusion, inversely correlated levels of miR-371a-5p and CPPED1 suggest a role for both in spontaneous delivery.

Understanding of timing of human parturition is incomplete. Therefore, we carried out proteomic analyses of full-term placentas from uncomplicated pregnancies to identify protein signatures associated with the onset of spontaneous delivery. We found quantitative associations of 10 proteins with spontaneous term birth, evident either in the basal or in the chorionic plates or in both. Additional 18 proteins were associated according to the location within placenta indicating local variations in protein amounts. Calcineurin-like phosphoesterase domain-containing 1 (CPPED1), a phosphatase previously suggested dephosphorylating AKT1/PKB, was one of the identified proteins. qRT-PCR revealed the mRNA level of CPPED1 was higher in elective caesarean deliveries than in spontaneous births, while immunohistochemistry showed CPPED1 in cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts. Noteworthy, phosphorylation status of AKT1 did not differ between placentas from elective caesarean and spontaneous deliveries. Additionally, analyses of samples from infants indicated that single-nucleotide polymorphisms rs11643593 and rs8048866 of CPPED1 were associated with duration of term pregnancy. Finally, post-transcriptional silencing of CPPED1 in cultured HTR8/SVneo cells by siRNAs affected gene expression in pathways associated with inflammation and blood vessel development. We postulate that functions regulated by CPPED1 in trophoblasts at choriodecidual interphase have a role in the induction of term labour, but it may be independent of AKT1.