Species Comparison of the Role of p38 MAP Kinase in the Female Reproductive System.

E 2 -ERα complex mediate cell proliferation via-p38-mediated mechanism. ERα nuclear receptors are only one of the key actors in the female reproductive system; non-genomic membrane ERs, upon E 2 binding, can also induce G-protein activation and MAPK pathways important to cell processes such as cellular proliferation. Infertility and lack of uterotrophic response has been reported in ERα knockout mice 57 . As previously noted, endometrial cell proliferation in the rat estrous cycle and NHP menstrual cycle is triggered by the release of E 2 from developing ovarian follicles. The ER is a zinc finger-containing transcription factor and member of the nuclear receptor superfamily with two isoforms, α and β, which are differentially expressed in a tissue-dependent manner in the uterus and ovary, respectively 58 . In the uterus, E 2 binds to ERα. ERα is then targeted to the nucleus where it stimulates the growth of normal and transformed endometrial cells of the female reproductive system ( Fig. 5 ). To facilitate this activity, ERα is localized in nuclei of uterine cells in a variety of species 11,59,60 . A. ERα expression in human uterus during the proliferative phase. Strong glandular (long arrow) epithelial nuclear expression. Note strong interstitial and moderate smooth muscle cell nuclear ER expression (arrowheads). B. ERα expression in rat uterus during estrus. Moderate glandular and luminal epithelial nuclear expression. C. ERα expression in non-human primate (Cynomolgus macaque) uterus during the proliferative phase. Strong endometrial stromal cell nuclear expression and moderate glandular epithelial nuclear staining. Immunohistochemical stain, original magnification ×10. Radi ZA, Khan NK, Toxicologic Pathology (34(4)), pp. 327–335, copyright 2006. Reprinted by Permission of SAGE Publications. Although ERα predominates over its ERβ analog, ERβ mRNA and ERβ protein are expressed in the nuclei of glandular epithelium in rats, macaques, and humans, suggesting a possible role for this isoform in modulating E 2 action 61,62 . Furthermore, nuclear receptors are not the only the key mediators in the female reproductive system. Non-genomic membrane ERs, upon E 2 binding, can also induce G-protein activation and MAPK pathways important to cellular processes ( Fig. 5 ) 63–65 . During the proliferative phase of the menstrual cycle, ERα mRNA and protein are expressed in all major uterine cells, including glandular epithelial, stromal, and uterine wall smooth muscle cells ( Fig. 6A ). Protein concentrations of both receptors decline during the secretory phase 59 . Similarly in the adult rat, ERα expression is lowest during the P and E phases, and consistent with cell proliferation patterns, ERα is only expressed in the glandular and luminal epithelia ( Fig. 6B ). ERα concentration rises significantly in these cells during the M phase in response to E 2 release 18,62 . In the luminal epithelial cells, proliferation continues to rise and although ERα levels drop, ERβ in uterine glandular epithelium of the rat is debatable 18,62 . In NHP uterine sections, strong endometrial stromal cell nuclear ERα staining, moderate to strong glandular epithelial nuclear staining, and mild luminal epithelial and myometrial smooth muscle cell nuclear staining were seen during the proliferative phase ( Fig. 6C ) 11 . During the secretory phase, mild glandular epithelial nuclear staining, negative myometrial smooth muscle cell and luminal epithelial nuclear staining, and strong endometrial stromal cell staining are observed. The changing pattern of E 2 and P 4 secretion during the NHP menstrual cycle is essential for the hormonal regulation of endometrial growth and differentiation and P action is essential for the proper maturation of the endometrium 47 . The transition from a proliferative (E 2 -dominated) to secretory (P 4 -dominated) endometrium results in the appropriate differentiation that permits implantation 47 . The numbers of ER in non-human primate endometrium are low when serum P 4 levels are elevated during the secretory phase of the menstrual cycle, but rise two to three fold when P4 levels decline during the proliferative phase 66 . In humans, extracellular signal-regulated kinase 1 (ERK1) is weakly expressed in glandular cells, but nearly undetectable in stromal cells of endometrial sections. ERK2 exhibited distinct glandular expression in both the proliferative and secretory phases, and a weak stromal cell expression 67 . The mechanism of E 2 cell growth stimulation in the uterus is mediated through the expression of a series of genes ( Fig. 5 ). Unlike other ligand-dependent receptors (e.g., glucocorticoid receptor), translocation back into the cytosol is not observed upon E 2 binding 62 , although it is interesting to note that other non-classical ligands (e.g., the ERα antagonist, ICI 182,780) can translocate ERα to the cytoplasm via a p38-mediated mechanism 68 . The ligand binding region of ERα has two transactivating domains, which are thought to act cooperatively. These are E 2 -independent activation function (AF1) and E 2 -dependent AF2 domain, located in the N- and C-terminal regions, respectively 69 . When E 2 binds to the AF2 region, a conformational change occurs that permits receptor binding to co-activating proteins (and co-repressors) that are necessary for transcription. Some of these co-activators include estrogen receptor-associated protein 160 (ERAP160), a splice variant of the progesterone receptor (PR) co-activator steroid receptor coactivator-1 (SRC-1) and member of the p160/SRC-type steroid receptor co-activators, and CBP/p300, a known co-activator of the nuclear receptor transcription factors cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and activating protein-1 (AP-1) 70 . The E 2 -independent AF1 domain is thought to be responsible for tissue and target specificity of the receptor molecule. AF1 activation, also leading to co-activator recruitment, is accomplished by MAPK phosphorylation. Importantly, direct phosphorylation and activation of ERα by p38 has been shown in human uterine endometrial cancer cells, suggesting that a similar mechanism is expected to play a role in normal uterine cell proliferation 69 . MAPKs have also been shown to phosphorylate ERα 71,72 and other nuclear hormone receptors containing AF1 and AF2 domains (e.g., peroxisomal proliferators activated receptor [PPAR-γ]) 73 , thereby modulating their activity. By binding to estrogen response elements (ERE), the nuclear E 2 -ERα transcription complex enhances the expression of genes (i.e., PR, OT, OTR) and protooncogenes (i.e., c-fos, jun [c-jun, jun-B and jun-D] and c-myc) 18,19 , regulates other non-ERE factors (i.e., epidermal growth factor (EGF), insulin-like growth factor (IGF-1) and their respective receptors, and cyclin D1, a first acting cyclin in cell cycle regulation and intra-cellular sensor of extracellular signals) 74 .

Rats are continuously polyestrus, with an average estrous cycle length of 4–5 days. The cycle is subdivided into four phases: proestrus (P), estrus (E), metestrus (M) (diestrus I), and diestrus II (D) 30,31 . The P phase begins when progesterone (P 4 ) levels decline as a result of luteolysis and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion from the anterior pituitary. In the adult female rat, the concentrations of the preovulatory estradiol (E 2 ), which is produced by ovarian follicles, peak during the P phase ( Fig. 1 ). As E 2 levels drop during the E phase, there is a corresponding lack of glandular and luminal epithelial growth and, in parallel, an increased apoptosis in these cells. Stromal cell proliferation is also seen 18 . The uterine lumen is dilated during both the P and E phases. As a result of the pre-ovulatory E 2 surge, ovulation and mating behavior occur at the beginning of the M phase. Notably, of the cell types comprising the uterus (stromal, epithelial, and myometrial), only the epithelial cells proliferate in response to E 2 in the adult rat and mouse. However, all cell types respond in the immature rodent 21 . P 4 , produced by the newly formed ovarian corpora lutea, also begins to rise and reaches a maximum peak during the longest phase of the cycle: the D phase. During the D phase, epithelial cell division and maturation is inhibited, but due to lowered levels of P 4 in the rat, typical endometrial gland secretion is not observed. Histological appearance of the rat uterus during proestrus (A), estrus (B), and metestrus (C). Smooth muscle hypertrophy (short arrows), endometrial stromal cell proliferation, stromal edema, and few mitotic figures are present during proestrus (A) . In estrus (B) , the uterine lumen is lined by very large, tall columnar epithelium, there is myometrial hypertrophy, and many of the luminal and glandular epithelial cells undergo vacuolar degeneration and necrosis (long arrows). In metestrus (C) , the luminal lining epithelial cells are reduced in height, there is cytoplasmic vacuolation of luminal epithelial cells (arrowheads), the stroma becomes denser, and mitotic activity decreases. (A) p38 expression in human uterus during the secretory phase. No epithelial p38 expression. (B) p38 expression in nonhuman primate (Cynomolgus macaque) uterus during the proliferative phase. Strong glandular epithelial cytoplasmic expression. (C) p38 expression in nonhuman primate (Cynomolgus macaque) uterus during the secretory phase. No epithelial expression. (D) p38 expression in rat uterus during proestrus. Strong glandular and luminal epithelial cytoplasmic expression. (E) p38 expression in rat uterus during estrus. Strong glandular and luminal epithelial cytoplasmic expression. Note strong myometrial smooth muscle cells cytoplasmic staining. (F) p38 expression in rat uterus during metestrus. Strong glandular and luminal epithelial cytoplasmic expression. Immunohistochemical stain, original magnification ×10. Radi ZA, Khan NK, Toxicologic Pathology (34(4)), pp. 327–335, copyright 2006. Reprinted by Permission of SAGE Publications. The histologic appearance of the uterus varies with the stage of the reproductive cycle. The P phase is characterized by distention of the uterine lumen with clear fluid and the lumen is usually lined by large low columnar cells 31 . Smooth muscle hypertrophy, endometrial stromal cell proliferation, stromal edema, proliferation of luminal and glandular epithelium, and a few mitotic figures are present during proestrus ( Fig. 3A ). In the E phase, the uterine lumen is lined by very large, tall columnar epithelium. There is myometrial hypertrophy, and many of the luminal and glandular epithelial cells undergo vacuolar degeneration and necrosis ( Fig. 3B ) 30,31 . In the M phase, the epithelial cells of the luminal lining are reduced in height, the stroma becomes denser, and mitotic activity decreases ( Fig. 3C ) 30,31 . During the D phase, the uterus is quiescent and appears shrunken with dense endometrial stroma, atrophied myometrium, very small lumen, and cuboidal glandular and luminal epithelium. Studies in several species have addressed the role of P 4 in the modulation of E 2 activity and maintenance of the uterus in a state of quiescence or inactivity 23–35 . P 4 is secreted for only a limited time by the rat, unless a leuteotropic signal from the pituitary is received 36 . In rats, that signal is prolactin, which is released upon cervical stimulation (copulation) 37 . With a new release of pre-implantation E 2 from the ovary, P 4 stimulates glandular secretion, endometrial stromal cell proliferation, and myometrial transformation. Furthermore, luminal epithelial cells undergo differentiation while preparing to receive the blastocyst. Interestingly, in the absence of pre-implantation E 2 in pregnant rats at this time, P 4 maintains the uterus in a neutral phase and the blastocyst in dormancy. Co-activators and repressors of steroid receptors govern appropriate E 2 /P 4 synergism 38 . For example, estrogen receptor (ER) negative uteri are hypoplastic, while P 4 receptor negative uteri are hyperplastic. This stringent regulation is required for appropriate uterine receptivity and embryo implantation in many species 39,40 . MAPK signaling pathways are activated during implantation in the rat 41 . Uterine receptivity, also known as the window of implantation, is identified by loss of progesterone ( Pgr ) gene expression from the epithelia 42 and by expression of a number of extracellular matrix molecules (e.g., secreted phosphoprotein 1 or osteopontin) and integrin heterodimers (e.g. αVβ3) that also initiate p38 MAPK signaling 40,43 . If no embryo implantation follows, the uterine endometrium, via an oxytocin (OT)-mediated mechanism, produces excess prostaglandin (PG), particularly PGF 2α , which is received by the ovary. As a result, P 4 production is decreased by the ovary and luteolysis occurs, marking the end of the D phase.

A variety of intracellular signals, including those involving ERα, c-fos, and p38α, orchestrate physiological events in the uterus during the secretory and proliferative phases of the estrous cycle in humans and NHPs. It is suggested that p38 has a modulatory role on human endometrial stromal cell proliferation and differentiation 81 . It is known that MAPKs play a role in regulating cellular hypertrophy and hyperplasia via mechanical stretch of the uterus 15 . Therefore, p38 inhibitors could play a role in various uterine and cervical proliferative conditions and cancers, including the pathophysiology of endometriosis 24,110 , endometrial, mammary, and ovarian cancers 111–113 , leiomyoma, and uterine fibroids 114,115 . MAPKs are known to regulate COX-2 expression and therefore, cervical cancers 116 .

There are several benefits to using the NHP, in particular Old-World monkeys, in female reproduction studies 44,45 . In general, NHP reproductive uterine and ovarian cycles and circulating steroid binding proteins, resemble those in humans, although other aspects (i.e., cycle length, gestation length) are different ( Table 1 ). Our discussion draws upon both human and NHP studies, with species differences noted. For purposes of this review, NHP means Old-World monkey of the subfamily Cercopithecinae (macaques, baboons and their phylogenetic kin). While the estrous and menstrual cycles have numerous similarities in humans and NHPs, a few primary differences or conventions should be noted ( Table 1 ). In contrast to the estrous cycle, which begins and ends at E phase and/or ovulation, the menstrual cycle begins and ends at menses, with ovulation occurring mid-cycle. Also, while the estrous cycle uses follicular (P and E phases) and luteal (M and D phases) phases, the menstrual cycle is most commonly divided into proliferative (follicular) and secretory (luteal) phases, which describes endometrial thickness 46 . Note that the proliferative phase occurs prior to ovulation in the menstrual cycle, although further proliferation does take place at the beginning of the secretory phase. Finally, humans and NHPs slough their endometrium when conception does not occur, whereas in other mammals it is reabsorbed. During the proliferative phase the endometrium begins to thicken and growth of all endometrial cells (endothelial, myometrial and stromal) takes place ( Fig. 2 ). Mitoses are present in the endometrial glandular epithelium during the follicular (“proliferative”) phase of the cycle, and in the stroma during the early luteal (“secretory”) phase. No evidence of mucus secretion or vacuolation is present during the proliferative phase. In the secretory phase, the endometrium slows its growth, stromal edema is evident, basal sub-nuclear secretory vacuoles are present in the glandular epithelium, and there is secretory exhaustion. Stromal edema is usually present at two times in the menstrual cycle, once in the mid-follicular/proliferative phase and once in the mid-luteal/secretory phase 11 . However, the menstrual and estrous cycles have more similarities. During the follicular phase paralleling the P and E phases, follicle stimulating hormone (FSH) and luteinizing hormone (LH) are released from the anterior pituitary under hypothalamic control, leading to ovarian follicular recruitment for ovulation and increased E 2 production. The endometrium thickens and P 4 levels are minimal. The mature ovarian follicle eventually secretes sufficient E 2 to promote a LH surge, which culminates in ovulation. Growth of all endometrial cells (endothelial, myometrial and stromal) takes place in response to this E 2 stimulation. P 4 levels increase in the stroma and remain elevated throughout the secretory phase, while levels in the epithelium decrease. The ruptured ovarian follicle develops into the corpus luteum and secretes P 4 and E 2 during the secretory phase. P 4 suppresses E 2 proliferation and causes a shift in proliferative activity to the stromal cells, which triggers epithelial cell differentiation in preparation for implantation. The changing pattern of E 2 and P 4 secretion during the NHP menstrual cycle is essential for the hormonal regulation of endometrial growth and differentiation, and P 4 action is essential for the proper maturation of the endometrium 47 . The transition from a proliferative (E 2 -dominated) to secretory (P 4 -dominated) endometrium results in the appropriate differentiation that permits implantation 47 . In NHP, P 4 primes the stromal cells to respond to pre-implantation (nidatory) E 2 for decidualization in early pregnancy. Stromal cell proliferation during the menstrual cycle is reported into the early to mid-secretory phase, when the endometrium reaches maximum thickness. Unlike the rat, P 4 levels in humans and NHPs produce glandular secretory activity. A pre-decidualization process, manifested by stromal edema, can be seen, as early as 10 days following the LH surge in humans. The reaction is characterized by eosinophilic and enlarged stromal cells that begin to surround prominent spiral arteries. Over the subsequent 3–4 days, this reaction spreads to the upper two thirds of the endometrium 48 , preparing a suitable environment for embryo attachment, successful implantation, and protection from invasive trophoblasts of the mother. Notably, pre-decidualization is part of the normal human menstrual cycle and the late luteal phase of macaque endometrium show decidual changes 48,49 . In primates, the ovarian cycle is uterine independent whereas it is uterine dependent in subprimate mammals. Luteolysis is driven by the withdrawal of the ovarian steroids, E 2 and P 4 , and release of PGF 2α . PGF 2α is released by the uterus in rats and other mammals, and the ovaries in NHPs 37 . Mediation of this luteolytic event by OT, although controversial, is reported in the NHP and also thought to be important in the rat 37,50 , but appears unlikely to play a role in human menses 48,49 . Cytokines, proteases, and PGs also play a role in menses 51,52 .

P 4 maintains the myometrium of the uterus in a state of quiescence during pregnancy by: 1) inhibiting the expression of contraction associated proteins (CAPs), which include connexin 43 and OTR 108 and 2) controlling PGF 2α , in part, via modulation of PG dehydrogenase and COX activity 109 . These possible roles for p38 have been previously discussed. IL-β contributes to parturition, by stimulating the production of PGF 2α via a COX-2-mediated mechanism. As in the normal cycle, PGF 2α induces contraction and luteolysis. Takanami-Ohnishi et al . have shown, using human decidual stromal cells, that this process is mediated by p38 kinase 22 . Both COX-1 and COX-2 are differentially expressed in the rat uterus, specifically in the epithelial and myometrial cells, during the estrous cycle, increasing dramatically during parturition and pregnancy 104 . Markedly up-regulated p38 MAPK activity has also been demonstrated in the uterus of term-pregnant non-laboring and spontaneously laboring women.

Much of our knowledge of the female reproductive cycle is drawn from research conducted on a variety of species. The rat, dog, and monkey are common laboratory animals used for testing new drug candidates developed for use in human medicine. However, there are significant differences among these preclinical species in the female reproductive system. Such differences should be taken into consideration by toxicologists and toxicologic pathologists when assessing the effects of new pharmacologic agents on the female reproductive system. Rat estrous cycle and differences in p38, c-fos and ER expression during various stages of the cycle. The average estrous cycle length in rats is 4–5 days. The cycle is subdivided into four phases: proestrus, estrus, metestrus, and diestrus. The proestrus phase begins when progesterone (P 4 ) levels decline as a result of luteolysis and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion from the anterior pituitary. In the adult female rat, preovulatory estradiol (E 2 ), which is produced by ovarian follicles, concentration peaks during the proestrus phase. As a result of the pre-ovulatory E 2 surge, ovulation occur during the estrus phase. In proestrus, endometrial stromal cell proliferation, stromal edema, and proliferation of luminal and glandular epithelium with subsequent increase in endometrial thickness take place. Myometrial hypertrophy also is seen during estrus. In the metestrus and diestrus phases, the luminal lining epithelial cells are reduced in height, the stroma becomes denser, and the uterus becomes quiescent with subsequent decreases in endometrial thickness. Overall, p38 strongly is upregulated during proestrus, estrus, and metestrus phases. c-fos is strongly upregulated in proestrus and metestrus and mildly upregulated in estrus. ERα expression is highest during proestrus and lowest in metestrus 11,30,31,119 . Human menstrual cycle and differences in p38, c-fos and ER expression during various stages of the cycle. The menstrual cycle begins and ends at menses, with ovulation occurring mid-cycle. The menstrual cycle is divided into proliferative (follicular) and secretory (luteal) phases, which describes endometrium thickness. During the follicular phase, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release from the anterior pituitary under hypothalamic control, takes place and leads to ovarian follicular recruitment for ovulation and increased estradiol (E 2 ) production. The endometrium thickens and progesterone (P 4 ) levels are minimal. The mature ovarian follicle eventually secretes sufficient E 2 to promote a LH surge, which culminates in ovulation. Growth of all endometrial cells (endothelium, myometrium and stroma) takes place in response to this E 2 stimulation. During the proliferative phase, the endometrium begins to thicken and growth of all endometrial cells takes place. In the secretory phase, the endometrium slows its growth. Overall, p38, c-fos, and ERα are strongly upregulated during the proliferative phase and lack expression in the secretory phase 11,53,117 . We first introduce these species differences in uterine cyclicity. The mechanism of early pregnancy trophoblast invasion is similar in rodents and the NHPs. The rat plays a prominent role in female reproduction research, making it a good model to detail the mammalian estrous cycle. There are significant interspecies differences in uterine cyclicity that may influence uterine functions and modulate the signaling pathways of MAPKs, c-fos, c-jun, and/or ER. For example, rats exhibit cyclic changes in the expression of c-fos and ERα 11 . The comparative aspects of the estrous cycle in the rat and dog and menstrual cycle in NHPs and human are summarized in Table 1 and Figs. 1 and 2 .

Depending on the tissue type, the nuclear protooncogenes are key players in cell proliferation, differentiation, and tumorigenesis, due in part to the direct effect of E 2 on these genes and their direct regulation by p38 MAPK 75 . E 2 -ERα transcription products, c-Fos and c-Jun, are members of the AP-1 transcription factor complex. Because increased expression occurs early in E 2 signaling, these genes are called “immediate early genes”. Notably, and as it will be discussed, there are differences in protooncogene expression between primates and other species during the menstrual cycle. Following E 2 treatment in mice, c-fos expression is observed only in the glandular and luminal epithelia, signifying a cell specific proliferative role 76 . Mendoza et al . further detailed c-fos expression in the rat, reporting a rise and peak in c-fos mRNA in both the glandular and luminal epithelia during the M phase, with a drop in concentration in both cells during the D phase. While c-fos protein concentrations rise during the M phase, concentrations increase only slightly during the D phase 18 . The rate of c-fos gene expression parallels that of formation of the active nuclear E 2 -ERα complex 19 . Studies in p300/CBP-associated factor (PCAF)– /– /PCAF-B +/– knockout mice have pinpointed the involvement of E 2 -ERα co-activators p300/CBP and PCAF in mediating c-fos expression in normal uterine cell growth 76 . P 4 appears to have more effect on c-fos mRNA than on the c-fos protein, a finding also reflected in the mouse 75 . Therefore, it is suggested that c-fos may have a role in the implantation period. Similarly c-myc mRNA has been shown to rapidly increase in response to E 2 in the rat 19 , with more specific distribution details described in the mouse 77 . Following E 2 injection in ovariectomized mice, c-myc protein was detected in uterine luminal and glandular epithelial nuclei during the P phase. P 4 also increased the number of c-myc positive stromal cells. Moreover, c-myc was detected in the nuclei of luminal and glandular epithelial cells during proestrus and on days 1 and 2 of pregnancy. These results suggest c-myc is a potent stimulator of cell proliferation 19 . In further contrast to c-fos and c-myc, c-jun is repressed by the ER in the rat epithelium 20,21 . c-jun expression was decreased in the epithelium and became evident in the stromal and myometrial cells early after E 2 stimulation in ovariectomized mice 75 . This work suggests that other Jun proteins (i.e., Jun-B, Jun-D) and c-fos may be important in early epithelial cell proliferation in the rodent. As might be expected from animal model studies, E 2 stimulates c-fos mRNA expression in the human endometrial epithelium and stroma during the proliferative phase of the menstrual cycle and occasionally during the mid-secretory phase 78,79 . Additionally, ERα-dependent c-fos expression in decidual tissue during pregnancy is very low, paralleling the decline of ERα in these cells and providing further evidence on the relationship between c-fos and ERα. c-myc is also expressed in the human endometrium during the menstrual cycle. Unlike in the rat, c-jun expression is strongly detected in both the proliferative and secretory phases of the menstrual cycle in humans 79 . Bircan et al . showed, using immunohistochemistry, that c-jun expression occurs primarily in the proliferative phase of the menstrual cycle 80 , which was further supported by Hong et al . showing a correlation between growth in cultured endometrial stromal cells and activated c-jun expression 81 . AP-1 subunits are dimeric basic region-leucine zipper (bZIP) proteins that belong to the Jun (c-jun, Jun-B and Jun-D), Fos (c-fos, Fos-B, Fra-1 and Fra-2), ATF (ATFa, ATF-2 and ATF-3), Maf, and Jun dimerization partner (JDP) subfamilies and recognize either phorbol-myristate-acetate (TPA)-response elements (TREs) or cAMP response elements (CRE) of DNA. Both Fos and Jun can act independently as transcription factors, activating transcription of growth-promoting genes or inhibiting growth-repressing gene transcription 82 . AP-1 regulation of cell processes (i.e., proliferation, survival, differentiation) is dependent on dimer composition, cell stimulus, cell type, and cell environment. MAPKs, such as p38, contribute to AP-1 regulation by stabilizing the participating proteins through phosphorylation and regulating their induction 82,83 . For the latter, p38 has been shown to phosphorylate and activate cis -element binding proteins important for c-fos induction, including CREB or ATF (2), which occupy the CRE consensus sequence and ELK-1, a monomeric ternary complex factor (TCF). This TCF is recruited by the serum-response factor (SRF), the protein that recognizes the serum response element (SRE) in response to ultraviolet (UV) irradiation and interleukin-1 (IL-1) stimuli. It is therefore hypothesized that p38 may play a similar role in AP-1 regulation in uterine cell proliferation. Cell cycle regulation is carried out by cyclins that bind to cyclin-dependent kinases (cdks) or cdk inhibitors to regulate phosphorylation of the retinoblastoma protein (pRB) and cell cycle progression. Studies in the murine system have suggested that a central point of regulation involves E 2 -induced uterine epithelial cell proliferation by nuclear accumulation of Cyclin D1 and pRB activation via a PI3 kinase/AKT/GSK3β- mediated pathway, which is inhibited by P 4 84 . However, these studies also note the need for a parallel pathway to initially trigger DNA synthesis. Correspondingly, the c-jun protein product of E 2 -ERα transcription is known to promote DNA synthesis, or the S phase of the cell cycle, in cultured normal human endometrial glandular cells via binding to the AP-1 sequence, with subsequent activation of Cyclin D1 85 . Many other factors have been shown to play a role in Cyclin D1 regulation in other systems in response to the E 2 -ERα interaction, including CREB, ATF-2, c-fos and pS1 in breast cancer cells. These studies reveal a host of complex signalling pathways that are most likely triggered upon E 2 and P 4 stimulation. Several pathways can involve p38 MAPK activation.

Toxicologists and toxicologic pathologists need to consider species differences when evaluating the effects of new pharmacologic agents on the female reproductive system. Experiments conducted on different preclinical species are not strictly comparable to that of humans because of significant interspecies differences in the physiology of uterine cyclicity. p38 MAPK signal transduction pathways are interconnected with ER, c-fos, c-jun and jun kinase, and regulate various aspects of the mammalian estrous and menstrual cycles, early pregnancy, and parturition. p38 is involved in: 1) the OT-OTR-mediated events and luteolysis, 2) regulation of uterine tissue breakdown and regeneration, 3) E 2 -induced uterine cell proliferation and growth, 4) implantation and uterine receptivity, and 5) maintenance of uterine quiescence during pregnancy and onset of parturition. Finally, p38 MAPK signaling plays a role in various pathological conditions in the female reproductive tract.

The p38 mitogen activated protein kinases (MAPKs) are members of discrete signaling transduction pathways that play significant regulatory roles in a variety of biological processes including inflammation, cell differentiation, and cell growth 1–4 . By participating in phosphorylation cascades, p38 modulates the regulation and activity of several transcription factors (i.e., activating transcription factors-1 and -2 (ATF-1, ATF-2); the p53 tumor suppressor protein; and CCAAT/Enhancer Binding Proteins-beta (C/EBPβ) 5–8 . These transcription factors lead to cytokine production, cell growth, apoptosis, and other cellular processes. Therefore, because of p38’s involvement in inflammation, specific p38 inhibitors are under development as anti-inflammatory medicines 3,9 . p38 MAPKs are activated by dual phosphorylation at residues thr180 and tyr182. Some p38 inhibitors (i.e., SB203580) compete for the ATP binding pocket and inhibit direct enzymatic activation, while other inhibitors (i.e., BIRB796) stabilize a conformation that is unable to bind ATP 10 . p38 MAPK, along with a variety of intracellular signaling pathways such as Estrogen Receptor α (ERα), c-jun and c-fos, orchestrate physiological events in the uterus during the menstrual cycle in both humans and in preclinical species such as nonhuman primates (NHPs). Emerging data suggests that uterine function effects may be attributable to the close interrelationships of these signaling pathways and their modulation 11 . c-fos and c-jun interact with the transcription factor activator protein-1 (AP-1) which translocates to the nucleus and binds to the AP-1 enhancer element to initiate a cascade of gene induction events that lead to cell proliferation 12 . MAPKs regulate AP-1 transcriptional activity and c-fos expression in the uterus and mediates mechanical stretch-induced c-fos expression in myometrial smooth muscle cells 13–16 . Estrogen stimulates DNA synthesis, and cellular proliferation and differentiation in the uterus of mammals 17,18 . Binding of estrogen to its receptor (ER) contributes to uterine cellular proliferation via increased expression of immediate early response genes 13,18–21 . It has also been suggested that the initial steps in the mechanism of mitogenesis by estrogen involve activation of c-fos gene expression in the rat uterus, emphasizing the orchestrated effort of these molecules in directing uterine function 19 . p38 MAPKs are thought to contribute to parturition 15,22,23 . Marked increases in the p38 kinase activity in the human uterus was observed on day 19 of gestation and during labor, and declined to the control levels post-delivery 23 . p38 MAPK has also been shown to be present in endometriotic cells from humans and activated by pro-inflammatory agents 24 . Moreover, various aspects of the mammalian estrous and menstrual cycles, as well as early pregnancy, involve regulatory roles for p38 MAPKs 5–8,25–28 , and p38 MAPK has been implicated in alterations and pathologies observed in the female reproductive system 1,12,29 . Considerable “cross talk” between the MAPK signaling pathways (e.g., p38, JUNK) may also play an important role throughout all stages of the female reproductive process. Collectively, these observations imply a close interrelationship between ER, c-fos, c-jun, and p38 in modulating uterine function during the estrous cycle and early embryonic processes. Therefore, pharmacologic modulation of one or more of these signaling molecules, coupled with interspecies differences in uterine cyclicity, may influence uterine function. This article provides a critical, comprehensive review of the known roles for p38 MAPKs and related pathways in the mammalian female reproductive system in of humans and in preclinical species, and signaling in reproductive pathophysiology. We first introduce fundamental differences and similarities in the mammalian reproductive system in humans and in preclinical species. We then explore in detail the known roles for p38 MAPKs pathways in female reproduction. With this foundation, we then provide a review of the pathological conditions in the female reproductive system under which p38 MAPKs may play a role.