Glycogen synthase kinase (GSK) 3 in Pregnancy and Parturition: a Systematic Review of Literature

Abstract

Introduction:

Human reproduction is a multifaceted process reliant on proper blastocyst implantation, placental and fetal membrane development, and delivery of a healthy baby. Multiple factors and pathways have been reported as critical machineries for cell differentiation and survival during pregnancy and most of them involve glycogen synthase kinase (GSK)3α for cell differentiation, survival and for maintaining cellular homeostasis. Several reports on GSK3’s functional role exist; however, a specific role of GSK3 in reproductive tissues and its contribution to normal or abnormal parturition are still unclear. To fill this knowledge gap, a systematic review of literature was conducted to better understand the functional role of GSK3 in various intrauterine tissues during implantation, pregnancy, and parturition.

Methods:

A systematic review of literature on GSK3 expression and function reported in reproductive tissues during pregnancy, published between 1980–2017 in English, using three electronic databases (Web of Science, Medline, and ClinicalTrials.gov) was conducted. The selection of studies, data extraction and quality assessment was performed in duplicate by two independent reviewers.

Results:

A total of 738 citations were identified, 80 were selected for full text evaluation and 25 were included for final review. GSK3 regulation and function was mostly studied in placental tissue and cells (12), cells of fetal origin (8), cells of uterine origin (6), and ovary (2). Measurements of total GSK3 and its isoforms (α and/or β) were determined mostly by western blot analysis. GSK3 is primarily reported as a downstream responder of AKT, Wnt and reactive oxygen species related pathways where is played a critical role in cell survival and growth in reproductive tissues.

Conclusions:

GSK3 is functionally linked to blastocyst implantation, establishment of pregnancy, trophoblast migration and invasion, decidualization, and term and preterm labor. Few reports specifically studied GSK3s expression and function in any reproductive tissues but mostly as secondary signaler of various conserved cell signaling pathways. Lack of scientific rigor in studying GSK3’s role in reproductive tissues makes this molecules function still obscure. No studies have reported GSK3 in cervix and very few reports exist in myometrium and decidua. GSK3 functions are hardly studied in reproductive tissues, and several knowledge gaps are identified requiring more functional studies in reproductive biology.

Introduction

Human reproduction is a complex process reliant on multitudes of cellular signaling molecules for proper implantation, development, and delivery of a healthy fetus. Understanding these regulatory molecules, their expression and function in various reproductive tissues are important to determine their contributions to various pregnancy related functions. Aberrant expressions and functions of these molecules can contribute to abnormal pregnancy environment and adverse outcomes (1–3). Glycogen synthase kinase (GSK) 3 is one of such key cell function regulatory molecule. GSK3 is a Serine/ Threonine kinase which is regulated by multiple phosphorylation sites (Fig. 1) and functions in a number of processes in the cell (4). GSK3 exists as two isoforms- the α (alpha) and the β (beta) with structural similarities within their kinase domain (5) and molecular weights of 51kd and 47kd respectively (6). Subtle differences arise in N- and C-terminal sequences (5) suggesting distinct biological functions(7).

Figure 1: Graphical representation of GSK3 isoform phosphorylation sites.

A) Showing the common regulatory sites of GSK3α/β. B) Representative structure of GSK3α containing a inhibition phosphorylation site (white) Ser21 on its N-terminal tail which can be phosphorylated by AKT/PI3K, mTOR/AKT, p90Rsk, PKC, PKA, and potentially the Wnt pathway. The N-terminal tail can act as a pre-phosphorylated substrate, or pseudosubstrate when stimulated by these upstream kinases. GSK3α also contains an over activation site in its kinase domain, Y279, which has been documented to be active by autophosphorylation. C) Representative structure of GSK3β containing a inhibition phosphorylation site (white) Ser9 on its N-terminal tail which can be phosphorylated by AKT/PI3K, mTOR/AKT, p90Rsk, PKC, PKA, and potentially the Wnt pathway. GSK3β also contains an over activation site in its kinase domain, Y216, which has been documented to be active by autophosphorulation. Uniquely, p38MAPK (T390) and ERK (T43) can also inhibit GSK3β by phosphorylating it on its C-terminus.

There are many upstream regulators of GSK3’s function in response to a variety of signals. Some of the main GSK3 regulators include Wnt, Phosphoinositide 3-kinase (PI3K)/ Protein kinase B (AKT), extracellular-signal-regulated kinase (ERK), and p38 mitogen activated kinase (MAPK). These pathways are reported to play important roles in pregnancy maintenance as well as term and preterm births (8–12). Close to 100 proteins have been suggested to serve as GSK3 substrates (13). GSK3 can be found in the cytosol, mitochondria, and nucleus (14) or bound to complexes such as the ‘β-catenin destruction complex’ (15) a molecule widely reported to promote cell survival. The ‘β-catenin destruction complex’ includes GSK3, adenomatous polyposis coli, Axin-1, casein kinase-1 (CK-1) and cytosolic β-catenin (16). In the absence of Wnt signaling, β-catenin gets phosphorylated by GSK-3, causing ubiquitin-mediated degradation of β-catenin that threatens cell survival (16).

p38MAPK is a multifunctional kinase that controls various cellular functions including cell growth and development and stress associated cell death. Recent reports from our laboratory (17, 18) and many others have noted stress kinases such as p38MAPK play a major role in regulating cellular functions in response to ROS during pregnancy and parturition. p38MAPK also has the ability to functionally control other primary and secondary signaling molecules like GSK3 via phosphorylation. Suggesting at term, active p38MAPK could phosphorylate (T390 (19, 20); Figure 1) and suppress GSK3. This inactivation can lead to an accumulation of β-catenin and the promotion of cell survival transcription factors. This increasing of β-catenin could play a homeostatic role between p38MAPK induced senescence and GSK3 regulated cell survival allowing intrauterine tissues to survive till delivery. However, due to large number of regulators that control GSK3’s function and target substrates for GSK3, the exact functional role of GSK3 is hard to determine.

A systematic review of literature was conducted to provide a comprehensive functional role of GSK3in reproductive tissues and to identify knowledge gaps to derive better hypothesis in future experiments. Our objectives are; 1) Identify reports on GSK3 associated functional changes in human and animal pregnancy and parturition, 2) Determine the mechanistic roles of GSK3 reported in human and animal pregnancy and parturition, and 3) Determine the knowledge gaps in GSK3’s functional role in human and animal pregnancy and parturition.

Methods:

A systematic review of the literature was conducted as per the requirements of the MOOSE group and PRISMA Statement (21, 22). This systematic review was registered in PROSPERO.

Search criteria for identification of studies

A systematic review of the literature published from 1980–2017 in English was collected from 3 databases: Ovid, Web of Science, and clinicaltrials.gov with the assistance of a librarian team at University of Texas Medical Branch in Galveston.

Search strategy

A search strategy was developed to study GSK3’s expression, function and mechanistic role in the intrauterine compartments and other reproductive organs of interest (ovaries, implanted blastocyst, fetal membranes, placenta, and uterus) during gestation (implantation through development) and parturition (at term or preterm delivery) (Appendix 1).

Selection criteria

Types of studies:

This review is restricted to studies primarily focusing on GSK3 in humans and all animal models of pregnancy. Original research studies were selected, which investigated GSK3 in various intrauterine components during implantation, pregnancy, and parturition. Studies were excluded if they were not related to GSK3, not related to reproductive period, specifically embryogenesis and contraception, review articles, below average quality score, or full text was not available (either published as an abstract only or not obtained from authors upon request). Studies were included if it reported samples from subjects at full term gestation (≥ 37 weeks) who were either in labor or not in labor at the time of collection. Studies were also included if results were solely related to patients with preterm labor and delivery (both spontaneous and induced), chorioamnionitis, or studies that utilized cell lines from gestational tissues irrespective of maternal age, fetal gender, socio demographic and other clinical factors, geographic location and ethnicity and race.

Types of outcome measures:

Two types of outcomes measurements reported were extracted for data analysis: 1) Expression and functional changes associate with GSK3 in reproductive tissues, and 2) Mechanistic roles of GSK3, during impanation, pregnancy, and parturition in human and animal models.

Specifically, we examined studies that assessed GSK3 RNA or protein expression changes, its activators and repressors, mechanisms of activation or inactivation and biological pathways impacted in response to changes in GSK3’s function.

Data collection and analysis

Selection of studies:

All citations retrieved through the search strategy were downloaded to a common storage folder where two authors (N.L. and L.R.) independently screened the title and abstracts of the citations. Titles and abstracts that were not related to GSK3, did not fit our inclusion criteria (i.e. reproductive tissues in implantation, pregnancy and parturition) or review articles were removed and duplicates were excluded. The studies, which fulfilled the selection criteria, were included for full text review and data extraction.

Data extraction:

A data extraction form was created for this study to collect the following information: study primary author, year of publication, journal of publication, country of origin for primary author, assessment of GSK3 isoforms, assay type used to determine GSK3 expression or function, gestational tissue studied, and the findings and or end phenotype measured in response GSK3s activity. Two authors (N.L. and L.R) independently extracted data from included reports, compared their findings, resolved disagreements through discussion and produced a single final form for each included study.

Quality assessment:

Quality assessment tool for systematic reviews have not been well defined for basic science research. Therefore, guidelines recently described by Hadley et al.(23) were used. Criteria used for quality improvement included the following; hypothesis driven study designs and approaches, strength of study design as defined by ascertainment of samples, description of sample collection, processing, and storage, description of reagents used for specific assays with detailed protocol that are sufficient to reproduce data, scientific rigor with details on appropriate controls and statistical tests, and sufficient explanation of results and if the results support the conclusions (Appendix 2) were analyzed per article and a score was given ranking them as poor, acceptable, or good quality.

Data synthesis

Based on our inclusion/exclusion criteria, data that reported GSK3’s role in tissues of interest (implanted blastocyst, fetal membranes, placenta, ovaries, and uterus) during implantation, pregnancy, and parturition either in humans or animal models were gathered. Based on these, we synthesized functions of GSK3 in each one of these tissues and document the major upstream regulators, downstream targets and functions, and project knowledge gaps that yet to be filled.

Results:

The search of 3 databases for reports published in English between 1980–2017 yielded 738 citations. After screening citations by title, 149 articles remained, which were again screened by abstract. A total of 80 studies were included for full text review. After removing duplicates, 59 studies remained. After full text review, 25 studies were included for final data extraction and analysis (Table 1) (Fig. 2).

Table 1:

Systematic Review Results

Authors	Country	Year	Species studied	Type of study	Type of sample	Methods	Upstream GSK3Î2	Form of GSK3	Down stream function
Hua et al.	China	2017	Human	Treatment of oligohydramnios	Amnion wish cells	WB and siRNA	Tanshinone IIA	P-GSK3Î2 S9	P-GSK3Î2 cant inhibit APQs which can now be upregulated
Lee et al.	United States	2016	Ewes	Establishment of pregancy	Oaries were collected and the CL isolated	WB	EGR->AKT	P-GSK3Î2 No phosphorilation site mentioned	Î2-Catenin and P-CREB, leads to programed survial and establishment of pregancy
Feng et al.	China	2016	Human	Magnetic fields (Other)	Amnion epithelial cells	WB	ROS	P-GSK3Î2 S9	Â
Astuti et al.	Japan	2015	Human	Trophoblast differentiation	HTR-8/Svnevo and human extravillous trophoblast	WB	PI3K and AKT	P-GSK3Î2 S9	Surivival
Lim et al.	Australia	2015	Human	Labor	Fetal membranes and myometirum	WB, inhibitor CHIR99021, and siRNA	TLRs	P-GSK3α/Î2 S21/S9	Inflammation, PGE2, Cox2, MMP9
Yung et al.	U.K	2014	Human	Tissue preservation (Other)	Placenta	WB	AKT-mTOR	P-GSK3α/Î2 S21/S9	Â
Zhou et al.	United States	2013	Human	Preeclampsia (PE)	PE placental cytotrophoblasts	WB	SEMA3B -> VEGF->PI3K/AKT	P-GSK3α/Î2 S21/S9	Î2-Catenin
O'Connell et al.	Australia	2013	Mice	Glucose transport	Placenta	PCR	Dexamethosone	Just total GSK3Î2	GYS1 and GBE1
Lassance et al.	Austria	2013	Human	Diabetes	Endothelial cells were isolated from third-trimester human placentas	Bio-Plex phosphoprotein detection kit and WB	PI3K/AKT	P-GSK3α/Î2 S21/S9	Î2-Catenin
Cheng et al.	U.K	2013	Human	Gestational diabetes mellitus (GDM)	Endothelial cells-HUVEC (Human umbilical vein endothelial cells)	WB	ROS	P-GSK3Î2 Y216	Defects in NrF2 necular accumlation and its down stream targets NQO1, Bach1, GCLM, and xCT
Yen et al.	Taiwan	2017	Human	Decidualization	Human endometrium and term Decidual tissues	WB	ILK	P-GSK3Î2	Prevented differentation and cell survival
Roseweir et al.	Uk	2012	Human	Trophoblast differentiation and invation	First trimester extravillious trophoblasts (HTR8SVneo)	WB	Kisspeptin-10	P-GSK3Î2 S9	Î2-Catenin in cytoplasum and enhance the effects on migration
Fischer et al.	Germany	2011	Human	Differentation	Bewo	Human Phospho-MAPK Array	Gal-1 ->AKT	P-GSK3α/Î2 S21/S9	ELK1 leading to syncytium formation
Lague et al.	Canada	2010	Mice	Trophoblast invasion	Decidua	WB	PI3K/AKT	P-GSK3Î2 S9	Represed decidua cells and limited transformation
Chiang et al.	Taiwan	2009	Human	PE	293T, BeWo, and JAR cells	IHC and WB	Hypoxia-> suppresion of PI3K/AKT singing	P-GSK3Î2 S9 and Y216	GMC1 degredation
Yung et al.	U.K	2008	Human	PE and IUGR	IUGR and PE placenta and JEG-3 cells	WB	AKT->mTOR	P-GSK3Î2 S9	eIF2BÎμ, 4E-BP1, and Eef2k
LaMarca et al.	United States	2008	Human	Trophoblast invasion	First trimester extravillous cytotrophoblast cell line-SGHPL-4	WB	EGFR-PI3K->AKT	P-GSK3Î2 S9	Î2-Catenin
Rider et al.	United States	2006	Rats(Ovariectomized)	Decidualization in early pregnancy	Uterine tissue (uterine horns from OVX rats)	Indirect immunoperoxidase analysis and WB	Wnt	P-GSK3Î2 S9	Î2-Catenin and TCF/LEF
Dash et al.	England	2005	Human	PE	Primary human extravillous cytotrophoblasts-SGHPL-4	WB	HGF->AKT	P-GSK3Î2 No phosphorilation site mentioned	Î2-Catenin
Boronkai et al.	Hungary	2005	Human	Syncitiotrophoblastic differentation	Placental tissue, umbilical cord, and membranes	WB	Phospho-AKT	P-GSK3Î2 No phosphorilation site mentioned	Â
Laviola et al.	Italy	2005	Human	IUGR	Placental tissue	WB	PI3K-AKT	P-GSK3α/Î2 S21/S9	Â
Bramer et al.	Canada	2017	Mares (Horse)	Glucose transport	Endometrial tissue	PCR	Just total GSK3Î2	Â	Â
Hou et al.	United States	2010	Bovine	Establishment of pregancy	Bovine luteal cells during early pregnancy	WB	LH	P-GSK3Î2 S9	mTOR phosphorilation of S6K1 and 4E-BP1
Kim et al.	United States	2008	Sheep	Trophoblast migration	Endometrial tissue,conceptus trophectoderm	WB	PI3K/AKT	P-GSK3Î2 S9	eIF2B
Sonderegger et al.	Austria	2010	Human	Invasion of human trophoblasts	Trophoblastic SGHPL-5 cells and primary extravillous trophoblasts	WB	Wnt	P-GSK3Î2 S9	Î2-Catenin

Figure 2: Prism flow chart.

Prism flow chart documenting GSK3 systematic review search strategy.

For our chosen timeframe the first GSK3 article was published in 2005 and reached the most number of publications noted in 2013 (Fig. 3 A). As expected, the reports evaluated were 99% basic science or research laboratory-based studies with only one paper related to a clinical case (24). In the latter report, high serum alkaline phosphatase in a 37 weeks pregnant patient revealed increased rate of cytotrophoblast proliferation due to inactivation of GSK3β downstream to AKT activation.

Figure 3: Systematic review quality assessment.

A) Quantity of GSK3 papers per year meeting our criteria. B) Percent of final articles from our systematic review that scored ‘good quality’ or ‘acceptable’. No papers were scored poor quality. C) Scores of each of our final 25 papers broken down per question.

Quality Assessment:

Twenty-one articles (84%) were graded as good quality, and the rest (4; 16%) were graded as acceptable quality, with none graded as poor (Fig. 3B–C). Good quality article provided adequate description of sample ascertainment, objectives of study, materials and methods, assay and analytical strategies and adequate description of data.

Main characteristics of studies:

The main characteristics and findings of the included studies are summarized in Table 1. GSK3 studies were conducted by researchers from many different institutions around the globe but predominantly from Europe (40%) and North America (32%). Reports included in our review investigated biological processes involved in pregnancy (starting from implantation) and parturition. Tissues and cells studied included from both fetal and maternal compartments.

Methods used for detecting GSK3 expression and function:

Mechanistic roles of GSK3 analyzed included determination of activators or pathways causing its activation. Functional changes of GSK3 were investigated as either pathways or the endpoint resulting from GSK3 activation. Most studies determined total GSK3 and its isoforms (α and/or β) expressions by western blot analysis. Total GSK3 expression were reported through qPCR (24, 25) and GSK3 activation was determined using indirect immunoperoxidase analysis (26). Phosphorylated forms of GSK3, specifically Ser21 for GSK3α and Ser 9 and Tyr 216 for GSK3β, were studied using western blot analysis, immunohistochemistry (27), indirect immunoperoxidase analysis (26), and also, by bio-plex phosphoprotein detection kit (1). To confirm the functional roles of GSK3 and to provide scientific rigor to their experimental approaches, many studies have included inhibitors of GSK3 like CHIR99021(28), LiCl (27), and siRNA (3) (Table 1).

Main findings:

Studies included in our final review reported multiple reproductive tissues derived from a number of different species. Among all the reports, 18 studies have used tissues/cells of human origin, 2 studies each in mice and sheep, and 1 study each using tissues/cells of rat, bovine and equine origin. We also found that 12 reports conducted studies using placental cells or tissues, 8 studies were using cells/tissues of fetal origin, 6 of maternal uterine origin, and 2 were from the ovary (Table 1).

Most GSK3 studies in reproductive tissues were conducted in placental tissues and cells indicating a role of GSK3 in trophoblast invasion and differentiation (24, 29–35). GSK3’s role in pathogenesis of certain disorders during pregnancy especially preeclampsia and Intra uterine growth restriction (IUGR) were also reported (27, 36–39). Studies of GSK3’s role in other tissues included the following; ovaries for establishment of pregnancy (8, 40), endometrium for decidualization (41, 42), and the fetal membranes and myometrium for initiation of labor (28). Interestingly, there were no reports of GSK3 in the cervix or the vagina.

GSK3 was reported to be involved in numerous signaling pathways in these reports. Thus, there were more than one upstream regulator and downstream targets for GSK3. The most common upstream regulator was Protein Kinase B also known as AKT. AKT was most commonly activated by Phosphatidylinositol-3 kinase(PI3K)(1, 29, 32–34, 39), but could also be activated by other less common activators like Galectin 1(43), hepatocyte growth factor (38), or was part of the AKT-mTOR pathway (mechanistic target of rapamycin)(37, 44) (Table 1). GSK3 was also shown to be a part of the Wnt or reactive oxygen species (ROS) signaling pathways (45, 46). GSK3 has multiple downstream targets that can lead to a number of different outcomes in reproductive tissues and cells. Eight studies reported β-catenin as downstream (1, 2, 8, 26, 30, 33, 35, 38) target of GSK3 promoting cell survival, while EIF2B (34, 37) was the second most common downstream target documented in 2 studies (Table 1) suggesting GSK3 mediates protein synthesis and cell proliferation.

Our review noted that GSK3 was studied as a part of a major signaling pathway either downstream or upstream to another molecule of interest and not necessarily as the primary molecule of interest for investigation. In placental tissues or cells, GSK3 was reported to be a part of the AKT pathway where it aids in trophoblast differentiation (24, 29, 31), trophoblast invasion (32–34) and likely in the pathogenesis of preeclampsia (2, 27, 37, 38) and IUGR (37, 39). GSK3 as a part of the Wnt signaling pathway was also shown to play a role in trophoblastic invasion using placental cells (35). Trophoblast migration was studied in endometrial tissues and conceptus trophectoderm where GSK3 was reported to aid AKT pathway by promoting translation via EIF2β (34). In ovarian tissues, AKT-GSK3 pathway was involved in pregnancy establishment (8).

As mentioned above, only a handful of studies examined GSK3 as their primary molecule of interest examining its functional role. Lim et al recently reported that in fetal membranes GSK3 may play major role in the terminal processes of human labor at term and preterm where GSK3’s increased activity leading to pro-labor and pro-inflammatory cytokines increase (28).

Astuti et al showed that recombinant Relaxin was able to induce proliferation and cell survival in HTR-8/SVneo cells (transformed extravillous trophoblast) via PI3K-AKT activation and GSK3β phosphorylation leading to cell survival (29). Similar results were reported by Roseweir et al in the same cell system where kisspeptin-10a and its receptor GPR54 regulated hypothalamic-pituitary-gonadal (HPG) axis to inhibit cancer metastasis. The report showed that a complex signaling pathway involving GSK3β might act as a negative regulator of trophoblast cell migration to possibly restrict the amount of invasion at the time of placentation (30).

Rider et al demonstrated that decidualization may be controlled by an endocrine dependent Wnt signaling where progesterone down regulates GSK3β via the Wnt pathway in uterine tissues (26) where estradiol caused nuclear translocation of β-catenin in the Wnt pathway. Also, in bovine luteal cells, Hou et al have shown that luteinizing hormone via phosphorylation of GSK3β amongst other mechanisms regulates progesterone synthesis, which is critical for the maintenance of pregnancy (40).

Hua et al reported that Tanshinone IIA, extracted from the root of Salvia Miltiorrhiza, stimulate aquaporin (AQP) expression (especially AQP8) in amnion Wish cells via phosphorylation of GSK3β and be a possible mode of treatment for oligohydramnios (3). Feng et al noted that human amnion epithelial cells increase mitochondrial permeability in response to an exposure to 50-Hz of magnetic field (45), an effect mediated by ROS/GSK3β signaling pathway (45). This frequency is important because it is emitted from most cell phones and computers. Other studies that studied about glucose transport in endometrial tissues (47) and placenta (25) and possible causes of gestational diabetes mellitus in HUVEC (Human Umbilical Vein Endothelial Cells) (46),placental tissue preservation (44) have also been included in the review.

Thus, the number of studies that look exclusively at GSK3’s mechanistic and functional role in reproductive tissues are few and far between.

Discussion:

Twenty-five studies reporting GSK3’s role during pregnancy and parturition were further analyzed for this review. Human and animal origin studies used tissues/cells from placenta, uterus, ovary as well as fetal origin (fetal membranes and Human Umbilical Vein Endothelial Cells) amongst others. AKT, Wnt, ROS signaling pathways commonly regulated GSK3 while β-catenin was the most commonly reported downstream target.

Based on our systematic review, we report that GSK3 is a major regulator of signaling pathways documented in a number of biological processes involved in pregnancy and parturition. These included establishment of pregnancy and blastocyst implantation, trophoblast migration and invasion, decidualization, placental glucose transport, complications of pregnancy such as: gestational diabetes mellitus, IUGR, preeclampsia, oligohydramnios and labor initiation at term and preterm amongst others. Each of these processes may be activated by different agents via separate signaling pathways that involve GSK3.The multitude of regulators, which phosphorylate and, in the process, modify the activity of GSK3 as well as a large number of possible substrates make understanding and pin pointing its exact function in a particular tissue quite challenging. Even though important biological processes during pregnancy and parturition have been studied, GSK3’s role in other important events like placental growth and its regulation under various oxidative conditions, remodeling of fetal membranes during pregnancy, cervical ripening, myometrial activation, etc. have not been reported.

GSK3 exists as two isoforms α and β (5). We found that in the studies included in our final review, most of them reported β isoform only, although the choice of β isoform for their studies were not properly rationalized. Only 6 of the studies reported and commented on both the isoforms (1, 28, 31, 36, 39, 44). It might be possible that GSK3β is the most studied form due a literature bias suggesting that mammalian GSK3β was more effective than GSK3α (48, 49). These reports; however, did not equalize the level of expression of the two isoforms hiding the fact that α and β are redundant in terms of regulating Wnt/β-catenin signaling (5).

GSK3 is a part of a number of signaling pathways in reproductive tissues. The upstream regulators may phosphorylate GSK3 at multiple sites –most commonly the Serine 21 for the α isoform and serine 9 and Tyr 216 for the β isoform. Only 2 studies looked at the Tyr 216 phosphorylation (27, 50) while 3 studies did not report specific phosphorylation sites examined in their experiments (8, 24, 38). Also, from studies conducted in other tissues, GSK3 was also phosphorylated at the Thr 390 residue in humans as part of the p38MAPK pathway (51). However, though p38MAPK was found to play a role in trophoblast migration [29], membrane senescence and parturition, IUGR development (39), and PE (24) none of the studies in this review examined this pathway or this particular phosphorylation site.

ROS induced p38MAPK is a signaling pathway noted to be active in reproductive tissues at term (17, 18). However, none of the studies in the review linked GSK3 and p38MAPK as a regulator of GSK3 function. Two of the studies showed that ROS (50, 52) can be an upstream regulator of GSK3. However, no connection between the two has been made in any of the studies that we reviewed. This might be an important link especially in determining the labor mechanism at term and preterm if looked into in future studies.

Following this review, we conclude that GSK3 is an important regulator that plays a major role in a variety of biological and metabolic processes in reproductive tissues. Targeting this molecule for therapeutic purposes in some of the obstetric complications discussed might prove to be fruitful. However, further studies need to be conducted to understand how activation or inactivation might affect other processes in these cells as it is part of a number of signaling pathways with possible cross talk between these pathways. Also, while conducting studies on GSK3 one needs to be extremely wary of which upstream regulators, phosphorylation sites, and downstream targets need to be looked at as that ultimately defines a particular action of GSK3.

Conclusion:

GSK3’s role in various utero placental tissues have been studies in connection with pregnancy and parturition. GSK3 studies are predominantly confirming its well reported activators or downstream effects. Novel conclusions cannot be derived other than what has already been reported in other biological system. This systematic review failed to find any unique role for GSK in association with the phenotypes of interest included. Function of GSK3 is dependent on the site-specific phosphorylation/modifications (activation/inhibition). Current literature is not sufficient to draw conclusions on activators and inhibitors of phosphorylation at specific sites. Pregnancy and parturition involve several unique changes, specifically constant changes in oxidative radicals and localized inflammation that are required to maintain tissue growth and integrity. GSK3’s role in maintaining tissue homeostasis during micro-environmental changes are hardly studied. Association between GSK3’s altered function contributing to pathological pregnancy outcomes are not reported and current reports are not convincing to draw a precise role due to lack of scientific rigor in experimental approaches. Thus, this review report several knowledge gaps. Mechanistic and functional studies are essential to better understand the contributions of critical cell function regulator. Understanding the role of GSK3 throughout gestation and in normal term pregnancies can provide insights into pathologic activation of its pathways that can cause adverse pregnancies.

Future Directions:

The systematic review has shown us that GSK3 is now emerging as a major regulator of biological funtions in reproductive tissues. However, studies that look primarily at GSK3 as the molecule of interest are few. Further studies in the field that help us understand the exact mechanistic role of GSK3 in reproductive tissues are needed. Such studies would help us understand if GSK3 might serve as a potential target for therapeutic interventions in conditions such as Preeclampsia, IUGR and preterm labor which are major causes of maternal and fetal mortality and morbidity.

List of abbreviations:

(GSK)3αβ

glycogen synthase kinase alpha or beta

AKT

Protein kinase B

PI3K

Phosphoinositide 3-kinase

ERK

Extracellular-signal-Regulated Kinase

P38MAPK

p38 Mitogen Activated Kinase

CK-1

Casein Kinase-1

IUGR

Intra Uterine Growth Restriction

mTOR

Mechanistic Target of Rapamycin

EIF2β

Eukaryotic Initiation Factor 2 (eIF2) β

HPG

Hypothalamic-Pituitary-Gonadal

HUVEC

Human Umbilical Vein Endothelial Cells

ROS

Reactive Oxygen Species

AQP

aquaporin