UNIVERSITATIS OULUENSIS MEDICA ACTA D D 1594 ACTA Ravindra Daddali OULU 2020 D 1594 Ravindra Daddali MOLECULAR MECHANISMS REGULATING THE ONSET OF LABOR COMPARATIVE PROTEOMICS AND miRNAOMICS OF HUMAN SPONTANEOUS AND ELECTIVE LABORS UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE; MEDICAL RESEARCH CENTER OULU; OULU UNIVERSITY HOSPITAL
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UNIVERSITY OF OULU P .O. Box 8000 F I -90014 UNIVERSITY OF OULU FINLAND
A C T A U N I V E R S I T A T I S O U L U E N S I S
University Lecturer Tuomo Glumoff
University Lecturer Santeri Palviainen
Postdoctoral researcher Jani Peräntie
University Lecturer Anne Tuomisto
University Lecturer Veli-Matti Ulvinen
Planning Director Pertti Tikkanen
Professor Jari Juga
University Lecturer Anu Soikkeli
University Lecturer Santeri Palviainen
Publications Editor Kirsti Nurkkala
ISBN 978-952-62-2805-1 (Paperback)ISBN 978-952-62-2806-8 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)
U N I V E R S I TAT I S O U L U E N S I S
MEDICA
ACTAD
D 1594
AC
TAR
avindra Daddali
OULU 2020
D 1594
Ravindra Daddali
MOLECULAR MECHANISMS REGULATING THE ONSETOF LABORCOMPARATIVE PROTEOMICS AND miRNAOMICS OF HUMAN SPONTANEOUS AND ELECTIVE LABORS
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU;OULU UNIVERSITY HOSPITAL
ACTA UNIVERS ITAT I S OULUENS I SD M e d i c a 1 5 9 4
RAVINDRA DADDALI
MOLECULAR MECHANISMS REGULATING THE ONSET OF LABORComparative proteomics and miRNAomics of human spontaneous and elective labors
Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defence inAuditorium F202 of the Faculty of Medicine (Aapistie 5 B),on 11 December 2020, at 12 noon
Supervised byProfessor Mika RämetProfessor Mikko HallmanDocent Antti Haapalainen
Reviewed byProfessor Markku VarjosaloDocent Tomi Airenne
ISBN 978-952-62-2805-1 (Paperback)ISBN 978-952-62-2806-8 (PDF)
ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)
Cover DesignRaimo Ahonen
PUNAMUSTATAMPERE 2020
OpponentProfessor Jaana Rysä
Daddali, Ravindra, Molecular mechanisms regulating the onset of labor.Comparative proteomics and miRNAomics of human spontaneous and electivelaborsUniversity of Oulu Graduate School; University of Oulu, Faculty of Medicine; MedicalResearch Center Oulu; Oulu University HospitalActa Univ. Oul. D 1594, 2020University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
Human parturition is a complicated biological process and the molecular mechanisms regulating the onset oflabor are not well understood. Several studies propose that gene variants, proteins and miRNAs play a majorrole in maintaining pregnancy and aberrations in protein and miRNA levels may lead to pregnancycomplications. The levels of proteins and miRNAs could prove to be useful biomarkers for monitoring theonset of labor.
In this study, using proteomics and a genome analysis, we identified Calcineurin-like phosphoesterasedomain-containing protein (CPPED1), as a potential biomarker of labor. CPPED1 was found to bedownregulated in spontaneous term placentas compared with placentas from elective deliveries. The siRNAknockdown of CPPED1 in trophoblasts derived cells mostly affects pathways related to inflammation andangiogenesis, and these functions may be involved in the induction of labor or in maintaining pregnancy. Inbladder cancer cells, CPPED1 has been shown to dephosphorylate AKT1 on the Ser-473 residue and topromote apoptosis via the AKT/PI3K pathway. In our studies using cells of placental origin AKT1phosphorylation levels were unaffected by CPPED1 expression, whereas its silencing led to the upregulationof negative regulators of the PI3K pathway.
In a comparative miRNAomics screen of spontaneous term vs. elective term placentas, we identified 54differentially expressed miRNAs, out of which 23 miRNAs were upregulated and 31 were downregulated byat 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 CPPED1and 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 diagnosticbiomarker for labor.
In order to elucidate the functions of CPPED1 we used a protein microarray to identify proteins thatinteract with CPPED1 in vitro and obtained a list of 36 proteins that potentially bind CPPED1. The interactionof CPPED1 with PAK4 and PIK3R2 was confirmed by co-immunoprecipitation and investigation of thecellular localization by BiFC studies showed that CPPED1 and PAK4 are colocalized in the cytoplasm,whereas CPPED1-PIK3R2 complex on the cell membrane. Further, by a mass spectrometry analysis, we foundthat CPPED1 dephosphorylates PAK4 on five different serine residues. The functional importance of thesephosphorylation sites is still unknown. PIK3R2 phosphorylation was unaffected by CPPED1.
Based on our studies we propose that the levels of CPPED1 and miRNA-371a-5p in the maternalcirculation could serve as biomarkers for the prediction of labor. The functional role of CPPED1 on PIK3R2and PAK4 remains to be studied.
Keywords: AKT1, CPPED1, mass spectrometry, miR-371a-5p, miRNA, PAK4,PIK3R2
Daddali, Ravindra, Synnytykseen liittyvät molekyylimekanismit. Ihmisenspontaanin ja elektiivisen synnytyksen vertaileva proteomiikka ja miRNAomiikkaOulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical ResearchCenter Oulu; Oulun yliopistollinen sairaalaActa Univ. Oul. D 1594, 2020Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Ihmisen synnytyksen käynnistyminen ja synnytys ovat monimutkaisia biologisia prosesseja, joiden kaikkiataustalla olevia molekyylimekanismeja ei vielä tunneta. Useat tutkimukset osoittavat, että tietyillä proteii-neilla ja mikroRNA-molekyyleillä on tärkeä rooli raskauden ylläpitämisessä. Näiden proteiini- ja mikroR-NA-määrien poikkeamat voivat johtaa raskauskomplikaatioihin. Proteiinien ja mikroRNA:n määrien mittaa-mista voitaisiin siten käyttää mahdollisina raskauskomplikaatioiden ja synnytyksen biomarkkereina.
Tässä tutkimuksessa löysimme proteomiikan ja genomin laajuisen assosiaatiotutkimuksen (GWAS) avul-la entsyymin nimeltä Calcineurin-like phosphoesterase domain-containing protein 1 (CPPED1), joka mah-dollisesti voi toimia spontaanin synnytyksen biomarkkerina. CPPED1-tasot laskivat spontaaneissa synnytyk-sissä, kun niistä saatuja istukoita verrattiin keisarinleikkauksen kautta saatuihin istukkoihin. Kun CPPED1-geenin ilmeneminen hiljennettiin istukan trofoblastisoluissa, tulehduksiin ja verisuonten uudismuodostuk-seen liittyvät signalointireitit muuttuivat eniten. Näillä muutoksilla saattaa olla merkitystä raskauden kes-toon. Aikaisemmassa tutkimuksessa on osoitettu, että virtsarakon syöpäsoluissa CPPED1 poistaa fosfaatti-ryhmän AKT1-entsyymin seriini-473 aminohapolta. Tämän fosfaatin poistaminen johti ohjelmoituun solu-kuolemaan AKT/PI3K-signalointireitin kautta. Meidän omissa tutkimuksissamme AKT1-entsyymin fosfory-lointi ei muuttunut istukan soluissa vaikkakin CPPED1:n määrä muuttui. Sen sijaan, CPPED1-määrän laski-essa PI3K-signalointireittiä negatiivisesti säätelevien proteiinien määrät lisääntyivät istukasta peräisin ole-vassa solulinjassa.
Vertailevassa mikroRNAomiikassa, jossa vertailtiin alatiesynnytyksistä ja keisarinleikkauksista peräisinolevien istukoiden mikroRNA-molekyylejä, havaitsimme merkittäviä muutoksia 54 mikroRNA-molekyyli-en määrissä. Näistä 54 mikroRNA:stä 23 mikroRNA:n määrät nousivat ja 31 mikroRNA:n määrät laskivatspontaaneiden täysiaikaisten synnytysten istukoissa. Eräs mikroRNA, jonka määrä nousi spontaanin synny-tyksen istukassa, oli miR-371a-5p. Osoitimme, että miR-371a-5p sitoutuu CPPED1:n lähetti-RNA:n 3’UTR-alueelle, minkä seurauksena lähetti-RNA:n määrä laski. CPPED1- ja miR-371a-5p-tasot siis korreloivatkäänteisesti toisiinsa istukoissa, jotka ovat peräisin spontaaneista synnytyksistä. Näin ollen äitien verestämitatut CPPED1 ja miR-371a-5p määrät voisivat olla potentiaalinen diagnostinen biomarkkeri spontaaninsynnytyksen ennustamisessa.
CPPED1:n toimintojen selvittämiseksi käytimme proteiinimikrosirutekniikkaa tunnistaaksemme proteii-neja, jotka voisivat mahdollisesti olla vuorovaikutuksessa CPPED1:n kanssa. Kyseinen proteiinimikrosirusisälsi noin 75% ihmisen solun kaikista proteiineista. Proteiinimikrosirumenetelmällä CPPED1:n osoitettiinsitoutuvan 36 erilaiseen proteiiniin. Näistä kahden proteiinin, PAK4:n ja PIK3R2:n, vuorovaikutuksetCPPED1:n kanssa varmistettiin kahdella muulla menetelmällä. Immuunisaostuksen avulla osoitettiin, ettäCPPED1 muodosti tiiviin kompleksin sekä PAK4:n että PIK3R2:n kanssa. Kaksoisfluoresenssikomplement-timenetelmän avulla havaitsimme, että CPPED1 sijaitsi solun sisällä samoissa paikoissa kuin PAK4 jaPIK3R2. Lisäksi massaspektrometrianalyysillä osoitimme, että CPPED1 poisti fosfaattiryhmiä PAK4:n tie-tyistä seriiniaminohapoista; näiden fosforylointikohtien toiminnallinen merkitys PAK4:lle on vielä tuntema-ton. Siten CPPED1 merkitys PAK4:n ja PIK3R2:n toiminnoille vaatii vielä lisätutkimuksia.
This thesis is based on the following publications, which are referred to throughout
the text by their Roman numerals:
I Haapalainen AM, Karjalainen MK, Daddali R, Ohlmeier S, Anttonen J, Määttä TA, Salminen A, Mahlman M, Bergmann U, Mäkikallio K, Ojaniemi M, #Hallman M, #Rämet M. (2017). Expression of CPPED1 in human trophoblasts is associated with timing of term birth. J Cell Mol Med. 2018 Feb;22(2):968-981
II Daddali R, Ojaniemi M, Hallman M, Rämet M& Haapalainen AM. (2020). MicroRNA-371a-5p targets CPPED1, and its expression in human placenta is associated with spontaneous delivery. Plos One. 2020 PLoS One. 2020 Jun 10;15(6):e0234403
III #Haapalainen AM, #Daddali R, Hallman M, Rämet M. Human CPPED1 belongs to calcineurin-like metallophosphoesterase superfamily and dephosphorylates PI3K-AKT pathway component PAK4 (Submitted)
# Equal contribution
The contributions of the author, R. Daddali, to the work focused on molecular
biology, proteomics and miRNAomics. He did all cell culture experiments (I, II,
III), the siRNA knockdown of CPPED1 (I), confocal imaging of p-FOXO1and p-
FOXO3, miRNA isolation, miRNA transfections, luciferase assay and data analysis
(II). He carried out the experiments including cloning, protein purification, BiFC
assays, co-IP, in vitro activity assays, CD and data analysis (III).
14
15
Contents
Abstract
Tiivistelmä
Acknowledgements 9
Abbreviations 11
Original publications 13
Contents 15
1 Introduction 19
2 Review of the literature 21
2.1 Labor/delivery and the length of pregnancy ........................................... 21
2.1.1 Major complications during pregnancy and delivery ................... 23
2.1.2 Biomarkers for labor and pregnancy complications ..................... 24
2.2 Human Placenta ...................................................................................... 26
2.2.1 Placental development and functions ........................................... 27
2.2.2 Placental genes, proteins and miRNAs ......................................... 29
2.2.3 Placental abnormalities and pregnancy complications ................. 30
2.3 Proteomics and pregnancy ...................................................................... 30
2.3.1 Proteomic changes in the placenta during pregnancy ................... 31
2.3.2 Human placental proteomics of preterm birth .............................. 32
2.3.3 Proteins as biomarkers of preterm birth from various body
The center for disease control and prevention (CDC) has initiated an effort to
improve the health of both neonates and mothers and to reduce the number of
maternal mortalities.
The regulation of the pregnancy duration is still poorly understood. In most
cases the pregnancy ends at the onset of labor and subsequent delivery. Many
pregnancy complications take place during labor. Most complications are evident
when the labor is either premature or postmature. Problems can be caused either by
the mother, fetus or the placenta. A common complication is premature rupture of
the membranes before the onset of labor. Another typical complication of
pregnancy is preeclampsia, which is seen as symptoms in the mother (high blood
pressure, proteinuria). In most severe cases the mother develops convulsions due
to eclampsia.
Detachment of the placenta (abruptio) with or without labor is a serious
although rare complications causing severe fetal asphyxia. Further complications
include the abnormal position or abnormal presentation of the fetus. Multiple
pregnancies are also associated with excessive complications; one of them is
premature birth. Complications during labor include shoulder dystocia, nuchal cord
and a prolapsed umbilical cord. Amniotic fluid embolism and placenta accreta are
complications manifesting during labor and delivery. Representative complications
are listed in Table 1.
24
Table 1. Common complications arising during labor
Complication name Features/Causes Reference
Obstructed labor
Preeclampsia
Postpartum bleeding
Postpartum infections
Gestational diabetes
Neonatal infections
Perinatal asphyxia
Too small or deformed pelvis. Prolonged labor, failure of vaginal delivery High blood pressure, proteinuria and risk of seizures in pregnant women. Restriction of fetal growth
Uterine atony, uterine constriction,
retention of parts of placenta
Consequences of heavy blood loss
Puerperal fever
Common in miscarriages
High blood sugar, preeclampsia,
polycystic ovary syndrome (PCOS)
Infection obtained intranatally: Str.
agalactiae. E. coli, Listeria infections
Insufficient O2 supply during birth
Multiple organ damage, persistent brain
damage
(Neilson, Lavender, Quenby, &
Wray, 2003)
(Lambert, Brichant, Hartstein,
Bonhomme, & Dewandre, 2014
(Weeks, 2015)
(Loudon, 1998)
(McIntyre et al., 2019)
(Chan, Lee, Baqui, Tan, & Black,
2013)
(Aslam et al., 2014)
2.1.2 Biomarkers for labor and pregnancy complications
There has been a long quest to understand the outcome of pregnancy and the
associated complications. It would be helpful to develop accurate biomarkers for
the prediction of a specific disorder and eventually to provide effective treatment
and prevention of severe consequences. Screening for fetal aneuploidy in the first
trimester is a commonly used test for the prediction of chromosomal incongruity.
The development of multiparametric tests for aneuploidy using multiple
biomarkers in early pregnancy helps in the prognosis of also other pregnancy
complications such as preeclampsia, fetal growth restriction, gestational diabetes
and preterm births.
25
In fetal growth restriction (FGR), the fetus fails to grow according to the
duration of the pregnancy. FGR is detected by fetal ultrasound and confirmed at
birth. Growth restricted fetuses have a higher risk of metabolic and cardiovascular
diseases in postnatal life than do children born at term (Malhotra et al., 2019). FGR
can arise from placental maternal or fetal factors.
For instance, FGR in severe preeclapsia is associated with the poor
development of placental villae. Many biomarker tests are available for the
prediction of FGR due to placental insufficiency, which includes fetal nuchal
expression in spontaneous and elective term placentas (II)
To determine the variations in the levels of miRNA expression between
spontaneous term labor and elective term labor placentas, we used miRNAomics
of human placentas from spontaneous deliveries (n = 6) and elective births (n = 6).
miRNAomics revealed that 23 miRNAs were upregulated, whereas 31 miRNAs
were downregulated in spontaneous term labor compared to elective term placentas
(Table 1 in original article II). Among the identified differentially expressed
miRNAs, some belonged to placental specific clusters, in which miR-323b-3p is a
member of the C14MC cluster, while miR-371a-5p, miR-371b-3p, miR-372-3p,
miR-372-5p and miR-373-3p are the members of the miR-371-3 cluster, located
adjacent to the chromosome 19 miRNA cluster. The miRNA genes of the rest of the
differentially regulated miRNAs are in different chromosomes.
5.2.2 Comparison of human placental proteomics and miRNAomics
identifies a miR-371a-5 -CPPED1 pair (I, II)
In miRNAomics, we found 54 miRNAs which were differentially expressed
between placentas from elective and spontaneous deliveries. Further, we
investigated whether there was any overlap between the proteomics and predicted
66
targets of miRNAs and identified three miRNA–protein target matches in the basal
plate of the placenta: miR-371a-5p–CPPED1, miR-3614-3p–ACTB and miR-
6872-3p–ACTB. We found that in spontaneous term deliveries, miR-371a-5p was
upregulated, whereas its target CPPED1 was downregulated indicating a potential
association between CPPED1 and miR-371a-5p.
5.2.3 CPPED1 mRNA levels are decreased and miR371a-5p levels
increased during spontaneous delivery (II)
In our comparative proteomic analysis and mRNA quantification analysis, we
found a decrease in the expression of the CPPED1 protein and mRNA levels in
both the basal plate and chorionic plates of the placenta from spontaneous term
labor. Further, we continued our study to measure CPPED1 mRNA levels by a
quantitative PCR (qPCR) analysis in spontaneous (n = 20) and elective caesarean
(n = 33) and preterm delivered placentas from both the chorionic and basal plate
regions. We found that CPPED1 mRNA levels decreased (1.5-fold, p< 0.001) in
the chorionic plate of the placenta at spontaneous preterm birth, but we did not
observe the same in the basal plate (Figure 1 in article II). Using miRNAomics we
observed that miR-371a-5p levels increased by 1.5-fold (p =0.04) in the basal plate
of spontaneous term placentas (Figure 2 in article II) compared to those of preterm
deliveries. The inversely correlated expression levels of miR-371a-5p and CPPED1
suggests that miR-371a-5p might regulate the levels of CPPED1 post-
transcriptionally.
5.2.4 miR-371a-5p and miR-520d-5p bind to the 3′UTR of CPPED1
In order to validate the binding of miR-371a-5p to the 3′UTR of the CPPED1
mRNA, we used a dual luciferase reporter gene assay to study the post-
transcriptional regulation of target mRNAs in intact cells. In addition to the miR-
371a-5p of miR-371-3 cluster, we also analyzed two other miRNAs of the C19MC
cluster, miR-520d-5p and miR-524-5p for their binding to the UTR and
posttranslational regulation of CPPED1 (Figure 3 in article II).
Both whole (WT) and truncated (lacking the terminal 33 bps, Mut) 3′UTRs of
CPPED1 mRNAs were cloned into the pmiRGLO vector and transfected into HEK-
293T continuous cell line. The mutant construct lacked the seed regions of both
miR-520d-5p and miR-524-5p, while both constructs contained the seed regions
for miR-371a-5p binding. The luciferase reporter assay revealed a significant
67
decrease (p = 0.004) in the relative luciferase activity of cells co-transfected with
the WT construct and the miR-371a-5p mimic (WT/miR-371a-5p) (n = 6)
compared to those transfected with the WT construct and the miRNA mimic
negative control (WT/miR-neg) (n = 6). We observed a similar decrease when the
cells were co-transfected with the WT construct and the miR-520d-5p mimic
(WT/miR-520d-5p) (n = 6, p = 0.004) when compared to WT/miR-neg (n = 6).
Altogether, these results indicated that miR-371a-5p of the miR-371-3 cluster and
miR-520d-5p of the C19MC cluster negatively regulates CPPED1 expression
(Figure 3 in article II).
5.3 CPPED1 classification and its functional role
5.3.1 CPPED1 sequence alignment suggests a metallophosphotase
family protein (III)
Zhuo et al., reported that based on a sequence analysis, CPPED1 contains a PP2A
catalytic domain from amino acids 50 to 250. According to the classification of the
protein phosphatase superfamily, members of the PP2A protein family do not
require any metal ions for their catalytic functions. CPPED1 phosphatase activity
was abrogated by trifluoroperazine, which is a PP2B specific inhibitor that does not
affect PP2A members (D. X. Zhuo et al., 2013), indicating a possible error in
classification, and prompting us to investigate CPPED1 further. Based on previous
reports, we searched for homology sequences using NPS Blast, and showed that
purple acid phosphatases from plants and phosphoesterases from Mycobacterium
and enterobacterial members are the closest homologues of CPPED1 (Figure 1 and
2 in article III).
5.3.2 CPPED1 dephosphorylates AKT1 in vitro
Previously, Zhang et al. reported that CPPED1 dephosphorylates AKT1 at the Ser-
473 residue and prevents bladder cancer progression in a mouse model (D. X. Zhuo
et al., 2013). In contrast to this finding, in our studies p-AKT of Ser-473 levels
remained unchanged, irrespective of CPPED1 expression levels in different term
placentas (Haapalainen et al., 2018). Using recombinant CPPED1, we performed
an in vitro phosphatase assay essentially as described by Gao et al., to confirm
whether CPPED1 dephosphorylates AKT1 (T. Gao et al., 2005). In this assay, we
68
incubated 30 ng of AKT1 and 25 ng of CPPED1 in Tris-HCl pH 7.5 buffer
containing 5 mM Mn2+ according to the protocol and another set without metal ions.
We observed that CPPED1 dephosphorylates AKT1 in the presence of both Mn2+
(maximum activity) and Ca2+ (moderately active) but no effect was observed in the
presence of Mg2+ (Figure 3 in article III).
5.3.3 CPPED1 interacts with multiple proteins in an in vitro protein
microarray
HuProt protein microarray has vast number of applications which includes
autoantibody-screening, protein-protein interactions, protein posttranslational
modifications and analyzing the modulation of protein expression levels in health
and diseased conditions (Huang et al., 2012). To identify proteins, which might
interact with CPPED1, recombinant purified CPPED1 with both V5 and His tags
were sent to a protein microarray core facility at Cambridge, the UK. After analysis
of protein-protein interaction studies, we obtained a list of 36 proteins (Table 1 in
article III), which interact with CPPED1 in vitro. A further pathway analysis was
done using DAVID functional annotation studies of all proteins to identify the
biological pathways affected by these proteins. Positive signal transduction
pathways and insulin pathways (PI3K/AKT pathways) were enriched in the list
(Table 2 in Article III).
5.3.4 CPPED1 and binding protein interactions confirmed by co-IP
and a BiFC assay
After the pathway analysis, we chose PAK4 and PIK3R2 for further studies. In
order to confirm the interaction between CPPED1 and binding partners, we
performed a co-IP of PAK4 and PIK3R2 with CPPED1. HEK 293T cells were co-
transfected with pCMVFlag Tag-CPPED1 and c-Myc tagged interacting partners.
CPPED1 and PAK4, PIK3R2 co-transfected cell lysates were pulled down with the
c-Myc antibody and probed with the anti-CPPED1 antibody (Figure 4 in article III).
We further used a BiFC assay to confirm the interaction between CPPED1 and
its putative binding partners the PAK4 and PIK3R2 proteins. We observed that cells
transfected with CPPED1 and PIK3R2 showed a fluorescence signal in the cytosol
with high intensity spots at the cell membrane, whereas PAK4 and CPPED1 over-
expressing cells gave the strongest signal in the cytoplasm (Figure 5 in article III).
Previously, it has been reported that phosphorylation of PAK4 is essential for its
69
nuclear translocation and functions (Li et al., 2012). Our studies confirmed the
interaction of CPPED1 with PAK4, however the functional importance of this
interaction remains to be elucidated.
5.3.5 CPPED1 dephosphorylation activity on the PAK4 and PIK3R2
proteins
After corroborating the interactions of CPPED1 with PAK and PIK3R2, we further
investigated the effect of CPPED1 on the phosphorylation status of PAK4 and
PIK3R2 proteins by an in vitro phosphatase activity assay. After the activity assay,
we analyzed the total phosphorylation levels on both the protein peptides identified
after a mass spectrometry analysis (Figure S7 in article III). It was found that
CPPED1 dephosphorylates five different serine residues in PAK4 including Ser 104,
Ser 167, Ser 173, Ser 174, Ser 181 and Ser 195 amino acids (Figure 6 in article III).
Out of the five, three have not been reported so far and the importance of these
phosphorylation sites in the functional regulation of PAK4 is not known. In contrast,
we did not observe any changes in the phosphorylation levels of the PIK3R2 protein,
which is mainly regulated by tyrosine-based phosphorylation/dephosphorylations
mediated by phosphotyrosine kinases/phosphatases.
70
71
6 Discussion
The molecular mechanisms associated with the onset of human parturition are
incompletely understood. We carried out proteomic and miRNAomic analyses of
human placentas, comparing samples from spontaneous term and elective term
labors to identify the proteins and miRNAs involved in the onset of labor.
6.1 Proteomic changes in spontaneous term and elective term
placentas (I)
We compared the human placental proteomes after spontaneous and elective term
delivery and detected statistically significant differences in the levels of ten proteins
between spontaneous and elective term deliveries. The quantitative changes were
significant in either the basal or the chorionic plates or in both. When we performed
a pathway analysis, we found that the majority of the GO terms were linked to
either the immune system or to structural/cytoskeletal organization. Structural
molecule activity (ACTB, KRT8 and KRT19) represented the major molecular
function. In addition, A2M and SERPINB2 were in the same blood coagulation
pathway. CPPED1 was not associated with any of the enriched GO terms. As a
phosphatase, CPPED1 is supposed to be involved in blocking cell cycle progression,
promoting apoptosis and affecting glucose metabolism (Vaittinen et al., 2013; D.
X. Zhuo et al., 2013). In addition, among the genes encoding the ten proteins whose
expression differed in spontaneous and elective births, genetic variants of the
CPPED1 gene associated with the duration of term pregnancies.
6.1.1 Placenta has a unique set of proteins at the fetal and maternal
sides
There is only a limited number of studies on the differential mRNA and protein
expression in fetal (chorionic) and maternal tissues (Decidua basalis). Two
independent studies found oxytocin as well as oxytocin receptor (OTR) levels to
be higher in the decidua and the fetal regions, in proximity of the decidua compared
to distal regions of the fetus (Chibbar, Miller, & Mitchell, 1993; Takemura et al.,
1994). Another study, which involved in identifying the low molecular weight
proteins and peptides between the chorionic and basal plates of the same placenta
reported 16 species with significant differences between the two regions (Kedia,
Nichols, Thulin, & Graves, 2015). When we compared the proteomes of the
72
chorionic and basal plates, we identified 19 proteins differentially expressed
between the foetal (chorionic plate) and the maternal (basal plate) sides of placenta.
When we compared the list of proteins from our studies with the proteins which
were reported in different studies and listed in a systematic review by Kacerovsky
et al., we found several proteins in common such as ACTB, keratin type I,
cytoskeletal 19, SERPINB3, annexin A3, annexin A5, and isoform 4 of
tropomyosin a-1 chain.
6.1.2 CPPED1 expression levels and polymorphisms associated with
the length of gestation and spontaneous term birth
CPPED1 is a phosphoprotein phosphatase encoded on chromosome 16p13.12. This
protein has 3 isoforms. Isoform 1 is a full-length protein comprising 314 amino
acids, while variant 2 is shorter form lacking amino acid residues 97-238 and
variant 3 varies in its sequence from isoform 1 at residues 97-122 and lacks amino
acids 123-314. The importance of these variants is not known yet. A qPCR analysis
of CPPED1 showed lower expression in spontaneous term labor compared to
elective term placentas and two of its SNPs were shown to strongly correlate with
the length of gestation. These notions emphasize its role in the onset of labor.
Previous studies have shown that CPPED1 dephosphorylates AKT1 on Ser473 and
inhibits AKT1 activation. However, in our studies p-AKT levels remain unchanged
despite varying CPPED1 expression levels. It is known that AKT1 phosphorylates
FOXO1 and FOXO3 and inhibits FOXO transcription factor mediated gene
expression by recruiting FOXO from the nucleus to the cytoplasm. FOXO interacts
with the progesterone receptor and this complex binds to DNA and activates
decidual genes, which play a major role in implantation. In addition to p-AKT
levels we did not observe any changes in FOXO1 and FOXO3 and their
phosphorylation levels. Interestingly, siRNA knockdown of CPPED1 in HTR-8
cells leads to the upregulation of the PI3K pathway negative regulatory gene
expression levels such as phosphoinositide-3-kinase interacting protein 1 (PIK3IP1) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG) indicating that CPPED1 might be involved in the
regulation of the PI3K/AKT pathway via other proteins rather than AKT1. Hence,
further studies are needed to understand the cellular functions and mechanism of
CPPED1 action in regulating the PI3K pathways in detail.
73
6.2 The expression levels of miRNAs associate with the outcome
of labor (II)
In this study we compared the miRNA expression levels between term placentas
from spontaneous births and elective births without labor. We were able to identify
a total of 53 different miRNA expression levels when comparing spontaneous and
elective births. Further, we compared our miRNAomics data with proteomics data
to find out whether these miRNAs target any of the reported protein candidates.
Indeed, using qpCR we found that miRNA371a-5p targets CPPED1 and its levels
were upregulated in spontaneous term placentas vs electively delivered placentas.
6.2.1 miR-371a-5p
The regulation of miR-371-5p is an epigenetic process which is dependent on
methylation and histone deacetylation mechanisms. The role of miR371-3 in the
placenta and during pregnancy was not known and further studies were needed to
understand its role in the placenta. Zhao et al., found that miR-371a-5p levels are
upregulated in gestational trophoblastic neoplasia (GTN) as compared to complete
hydatidiform moles and the elevated levels are associated with enhanced
proliferation, differentiation and invasion in choriocarcinoma cells. This dentotes
that miR371-3 may serve as a diagnostic marker for GTN (J. R. Zhao et al., 2018).
Other pregnancy disorders, in which miR-371-5p is involved, are intrahepatic
cholestasis, where patients have elevated levels of miR-371-5p in their serum (Zou
et al., 2018). Recurrent pregnancy loss is a condition in which apoptosis dominates
trophoblast growth which results in an adverse pregnancy outcome. The expression
of miR-371-5p and its target gene X-linked inhibitor of apoptosis protein (XIAP)
are greatly reduced, which is associated with increased apoptosis which further
leads to pregnancy loss (Du et al., 2019). In our studies we discovered that the
levels of miR-371a-5p are higher in term placentas from spontaneous deliveries
compared to elective term placentas.
6.2.2 CPPED1 is targeted by miR-371a-5p and their expression levels
inversely associate with each other
In our previous studies we have shown that the expression level of CPPED1
associates with the timing of birth. In addition, spontaneous term placentas have
decreased levels of CPPED1 compared to elective term placentas. Our
74
miRNAomics data showed that miRNA-371a-5p levels are upregulated in term
placentas. Furthermore, we showed that miRNA-371a-5p targets CPPED1 in vitro
assay. Interestingly, we did not observe any changes in the levels of CPPED1 in
preterm delivered placentas compared to spontaneous term placentas. Quantifying
CPPED1, along with miR-371a-5p may reveal an association, which indicates the
length of pregnancy or the onset of labor.
6.3 Homo sapiens CPPED1 belongs to the calcineurin-like
metallophosphoesterase superfamily (III)
Previously, CPPED1 was categorized as a protein phosphatase 2B family member
(D. X. Zhuo et al., 2013). Our homology search using the NPS Blast tool suggested
that CPPED1 belongs to the metallophosphatase (PPM) superfamily. However,
interacts with PIK3R2 and the interaction was confirmed with co-
immunoprecipitation and BiFC studies. Further, mass spectrometry analysis of
78
PIK3R2 revealed that its phosphorylation levels are unaffected by CPPED1, but
the effect of CPPED1 on the expression levels of p85β is not known.
Fig. 10. Compex interplay of CPPED1 with different proteins of the PI3K/AKT and PAK4
signaling pathways. Binding of growth factors to tyrosine receptors leads to activation
of GRB2, which further activates PI3K via Ras GDP/GTP proteins. CPPED1 interacts
with GRB2. Activated PI3K converts PIP2 to PIP3 which recruits PDK1 and AKT1 to the
cell membrane. CPPED1 interacts with p85β which is a regulatory subunit of PI3K, but
the significance of this interaction is yet to be elucidated. PDK1, PDK2 and ILK
phosphorylate AKT1, which leads to the full activation of AKT1. Similarly, PP1, PP2A,
PHLPP dephosphorylate AKT1 which leads to AKT1 inactivation. CPPED1 is shown to
dephosphorylate AKT1 on Ser-473 and promote apoptosis. Activated AKT1
phosphorylates FOXO1/3 transcription factors leading to the nuclear exclusion of FOXO
proteins. On the other hand, the PAK4 protein is activated by the KRAS or PI3K protein,
which further activates AKT1 by phosphorylation. Both AKT1 and PAK4 reciprocally
regulate each other. PAK4 functions are regulated by phosphorylation/
dephosphorylation events. Autophosphorylation of PAK4 on Ser-474 leads to nuclear
translocation and activation of the expression of certain genes. CPPED1 interacts and
dephosphorylates several serine residues which might prevent nuclear translocation
and regulation of PAK4 functions are yet to be elucidated. In our studies we have shown
the interaction of CPPED1 with PAK4 and PIK3R2 which might regulate the PI3K
pathway at various stages either by p85β or PAK4 in addition to previously known AKT1
dephosphorylation (Modified based on Crivellaro et al., 2016; Thillai et al., 2017).
In a recent study, Zhuo et al., showed that the mechanism of CPPED1 tumor
suppressor activity in bladder cancer via decreasing IL-6 secretion which
79
inactivates the STAT3 signaling pathway. A gene expression analysis of CPPED1
overexpressing bladder cancer cells by microarray identified that IL-6 was
downregulated (D. Zhuo, Wu, Luo, Deng, & Niu, 2019) , whereas in contrast to
this, in our previous findings the knockdown of CPPED1 in HTR-8 trophoblast
cells led to a three-fold decrease in IL- 6 levels, implying that the function of
CPPED1 might be dependent on cell type and origin. It is interesting to note that
IL-6 reduces PIK3R2 levels by epigenetic modifications, whereas CPPED1
silencing leads to upregulation of IL-6 in bladder cancer. This suggests that
CPPED1 might influence p85β expression levels either directly or via upregulating
the levels of IL-6 in the cellular milieu. Further investigation is needed to
understand the connection between CPPED1, IL-6 and PIK3R2. This information
may help in the elucidation of the cancer progression.
6.4 Prospects for future studies
Using proteomics and miRNA-omic studies, we have identified different proteins
and variations in miRNA expression levels between term and elective term
placentas.
Our comparative proteomics studies have identified 10 proteins which were
differentially expressed in term placentas compared with elective term placentas.
Among the ten proteins, the following four were upregulated: ACTB, b-2-
microglobulin (B2M), keratin type II cytoskeletal 8 (KRT8) and 19 (KRT19),
whereas six proteins, including a-2-macroglobulin (A2M), CPPED1, cytochrome
b5 (CYB5A), haemoglobin subunit c-2 (HBG2), peroxiredoxin- 2 (PRDX2) and
plasminogen activator inhibitor 2 (SERPINB2) were downregulated and all
proteins showed differences in their expression at the chorionic (fetal side) and the
basal plate (maternal sides) of the placenta, suggesting a fetal - maternal gradient.
Further, we confirmed the associations of CPPED1 SNPs in term deliveries and
that its silencing in trophoblasts affects inflammation and angiogenesis. More
studies are needed with larger placental samples to confirm these changes and to
further strengthen the association of CPPED1 with the onset of labor.
Our miRNAomics demonstrated the differential expression levels of 54
miRNAs and we validated the significant differences in miR-371a-5p levels
between term and elective term placentas. Further, we characterized the connection
between miR-371a-5p and CPPED1 protein levels and they were found to be
inversely correlated in term placentas. Measuring the levels of both miR-371a-5p
80
and the CPPED1 protein during different gestational ages using more placentas
would reveal the kinetics in their variation and importance for the onset of labor.
CPPED1 is an unexplored protein of the protein phosphatase family and known
to play an important role in inhibiting bladder cancer progression via affecting the
AKT/PI3K pathway by dephosphorylating the Ser-473 residue on AKT1. We have
studied in vitro protein-protein interactions of CPPED1 and its binding partners
using the HuProt protein array and confirmed the interaction of CPPED1 with
PAK4 and PIK3R2. An in vitro phosphatase activity assay and mass spectroscopy
revealed that CPPED1 dephosphorylates PAK4 on several different serine residues.
Characterizing the importance of these phosphorylation events will help to
understand the functional regulation of PAK4. Furthermore, studying the effect of
CPPED1 on PIK3R2 expression levels will likely elucidate the functional
mechanism of CPPED1 in the cellular milieu.
81
7 Conclusions
Our comparative proteomics study of spontaneous and elective term labors
identified different protein profiles for the maternal and fetal sides of the placenta.
The levels of these proteins are altered in spontaneous labor. Previous studies
showed that the levels of CPPED1 are reduced with the progression of bladder
cancer and lower levels are associated with low survival, indicating its potential as
a biomarker for cancer progression. Similarly, we found lower levels of CPPED1
to be associated with spontaneous term birth.
miRNAomics were used to determine the differential miRNA expression
patterns between term and elective term phenotypes of labor. Measuring these
identified miRNAs between these two groups and identifying their functional and
biological roles in the placenta would contribute to understanding the roles of these
miRNAs in the onset of labor. In this study we have demonstrated that the
expression levels of a protein and miRNA pair, CPPED1-miR-371a-5p, showed a
significant difference between spontaneous term and elective term placentas and
measuring their levels could be used as a biomarker for labor.
We showed that CPPED1, a novel protein phosphatase dephosphorylates
AKT1 on the Ser-473 residue. However, AKT1 phosphorylation levels remain
unaffected despite of the variations in CPPED1 levels in different term placentas.
The siRNA knockdown of CPPED1 in HTR8 trophoblasts led to an increase in the
upregulation of negative regulators of the PI3K pathway, suggesting that CPPED1
might play a central orchestrating role in fine tuning of the AKT pathway via other
mechanisms if not by AKT1.
Finally, we characterized the protein interactome of CPPED1 using a protein
microarray and corroborated the interaction of CPPED1 with PAK4 and PIK3R2,
both involved in the regulation of the AKT/PI3K pathway. Based on proteomics,
genetics and epigenetics CPPED1 associates with spontaneous birth and may turn
out to be a specific biomarker for the onset of labor. CPPED1 might be a
pleotrophic protein which is involved in the regulation of different cellular
processes by altering the functions of the target protein by either posttranslational
modifications such as dephosphorylation, as seen in case of AKT1 and PAK4, or
by inhibiting or decreasing the levels of certain genes, as in the case of IL-6. Further
studies, identifying the significance of CPPED1 interactions with its binding
partners, might reveal the functional role of CPPED1 in the cellular milieu.
82
83
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Original publications
I Haapalainen AM, Karjalainen MK, Daddali R, Ohlmeier S, Anttonen J, Määttä TA, Salminen A, Mahlman M, Bergmann U, Mäkikallio K, Ojaniemi M, #Hallman M, #Rämet M. (2017). Expression of CPPED1 in human trophoblasts is associated with timing of term birth. J Cell Mol Med. 2018 Feb;22(2):968-981
II Daddali R, Ojaniemi M, Hallman M, Rämet M& Haapalainen AM. (2020). MicroRNA-371a-5p targets CPPED1, and its expression in human placenta is associated with spontaneous delivery. Plos One. 2020 PLoS One. 2020 Jun 10;15(6):e0234403
III #Haapalainen AM, #Daddali R, Hallman M, Rämet M. Human CPPED1 belongs to calcineurin-like metallophosphoesterase superfamily and dephosphorylates PI3K-AKT pathway component PAK4 (Submitted)
Reprinted (I and II) articles with the permission from corresponding journals.
Original publications are not included in the electronic version of the dissertation.
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MOLECULAR MECHANISMS REGULATING THE ONSETOF LABORCOMPARATIVE PROTEOMICS AND miRNAOMICS OF HUMAN SPONTANEOUS AND ELECTIVE LABORS
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