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Nuclear factor–kappaB: a main regulator of inflammation and cell survival in endometriosis pathophysiology
Nuclear factor–kappaB: a main regulator of inflammation and cell survival in endometriosis pathophysiology lez-Ramos, M.D., Ph.D.,a Sylvie Defre re, Ph.D.,b and Luigi Devoto, M.D.a Reinaldo Gonza a
Instituto de Investigaciones Materno Infantil, Departamento de Obstetricia y Ginecología, Hospital Clínico San n, Facultad de Medicina, Universidad de Chile, Santiago, Chile; and b Department of Gynecology, Universite Borja-Arriara Catholique de Louvain, Brussels, Belgium
Objective: To update, analyze, and summarize the literature concerning nuclear factor–kappaB (NF-kB) participation in endometriosis pathophysiology. Design: Review. Result(s): Nuclear factor–kappaB is physiologically activated in the human endometrium, showing variable activity. A cyclic p65DNA binding pattern was shown in the endometrium of healthy women. This cyclic pattern was altered in the endometrium of patients with endometriosis. Nuclear factor–kappaB is basally activated in peritoneal endometriotic lesions, showing higher p65 activity in red endometriotic lesions than in black lesions. In vivo and in vitro studies show up-regulation of inflammation and cell proliferation and down-regulation of apoptosis by NF-kB activity. Iron overload has been shown in the pelvic cavity of endometriosis patients, and iron overload and oxidative stress activate NF-kB in macrophages, which have been shown to participate in the endometriosis-associated inflammatory reaction. Conclusion(s): Nuclear factor–kappaB activation dysregulation in the endometrium of endometriosis patients may explain some endometrial biological alterations associated with endometriosis. The scientific evidence strongly suggests that NF-kB activity in endometriotic cells stimulates inflammation and cell proliferation and inhibits apoptosis, favoring the development and maintenance of endometriosis. Iron overload in the pelvic cavity of endometriosis patients could be a main factor enhancing oxidative stress and activating NF-kB in a chronic manner, contributing to endometriosis establishment and growth. Use your smartphone (Fertil SterilÒ 2012;98:520–8. Ó2012 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Apoptosis, cell proliferation, endometriosis, endometrium, inflammation, iron, and connect to the macrophages, nuclear factor–kappaB Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/gonzalez-ramosr-nuclear-factor-kappab-inflammation-cell-survival-inendometriosis/
T
he survival of endometrial cells on ectopic locations, named endometriosis, is a complex pathophysiological phenomenon. Numerous proinflammatory cytokines and growth factors have been involved in the development and maintenance of this disease (1–8). Most of the main proteins that have been implicated in
an endometriosis-associated proinflammatory environment are up-regulated by the transcription factor nuclear factor–kappaB (NF-kB). The proinflammatory peptides implied in this local event in the pelvic cavity of endometriosis patients contribute to stimulate cell proliferation and inhibit apoptosis of endometriotic cells,
Received May 18, 2012; revised June 5, 2012; accepted June 8, 2012; published online July 6, 2012. R.G.-R. has nothing to disclose. S.D. has nothing to disclose. L.D. has nothing to disclose. lez-Ramos, M.D., Ph.D., IDIMI, Facultad de Medicina Centro, UniverReprint requests: Reinaldo Gonza n, Av. Sta. Rosa 1234, 2 piso, Santiago, Chile sidad de Chile, Hospital Clínico San Borja Arriara (E-mail: [email protected]). Fertility and Sterility® Vol. 98, No. 3, September 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.06.021 520
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facilitating endometriotic cell survival (9–14). Nuclear factor–kappaB has been shown to be a key protein favoring cell proliferation and inhibiting apoptosis in various cell types, including endometrial and endometriotic cells (11, 13, 15–22). This review is focused on NF-kB– controlled gene transcription, NF-kB signaling, and interactions involved in endometriosis pathophysiology and particularly in endometriosis-related inflammatory process and endometriotic cell survival. In addition, because iron overload in the pelvic cavity of endometriosis patients has been identified VOL. 98 NO. 3 / SEPTEMBER 2012
Fertility and Sterility® as a possible mechanism at the origin of endometriosis, and evidence points to iron-mediated oxidative stress as an NFkB activating factor, the literature concerning iron overload in endometriosis and iron overload–dependent NF-kB activation will be examined as a possible factor contributing to the pathogenesis of endometriosis.
FIGURE 1
NF-kB The transcription factor NF-kB is a family of peptides formed by five subunits: RelA or p65, RelB, c-Rel, p50/p105, and p52/ p100. These subunits bind forming different NF-kB dimers that remain inactive by their union to the NF-kB inhibitory protein IkB. This protein prevents NF-kB–DNA interaction, and thus NF-kB target genes transcription is blocked (23–29). The canonical NF-kB pathway is activated by proinflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1b and by lipopolysaccharide (LPS) derived from bacteria that stimulate the I-kappaB kinase complex (IKK), phosphorylating IkB. IkB phosphorylation and subsequent ubiquitination allow its degradation by the proteasome. As a result, NF-kB free dimers are able to bind to DNA, initiating NF-kB target genes transcription. This pathway activates mainly p50/p65 NF-kB dimers that have been involved in the transcription of genes that regulate innate immunity, inflammation, and cell survival. Atypical pathways activating also p50/p65 NF-kB dimers via tyrosine kinase (TyrK) or casein kinase-II (CK2) are triggered by stimuli such as hypoxia, oxidative stress (which can be caused or favored by iron overload), and ultraviolet radiations (Fig. 1). Other important NF-kB dimers regulating gene transcription are p50/p50 dimers, which lack the transcriptional activation domain but bind to DNA because they have the Rel homology domain like all NF-kB subunits. Thus p50/p50 dimers are transcriptionally inactive, and consequently they have been linked to inflammation ceasing. Nuclear factor–kappaB p52/RelB dimers are activated by alternative pathways and they have been associated with lymphoid organogenesis, B-cell maturation, and humoral immunity (16, 29, 30). Research on NF-kB and endometriosis has been carried out mainly considering the canonical or atypical activation pathways, which lead to the activation of p50/p65 dimers, and this paper relates consequently.
PHYSIOLOGIC ACTIVATION OF NF-kB IN THE HUMAN ENDOMETRIUM Nuclear factor–kappaB p50 and p65 subunits, IkB and IKK are expressed in human endometrial stromal and epithelial cells (31–33). The expression of p65 is higher in the endometrial epithelium than in the stroma (34), which could mean that the canonical and/or atypical NF-kB pathways are physiologically more important in the endometrial epithelium than in the stroma. This seems logical, because the endometrial epithelium is the first defensive barrier in case of infections, and NF-kB p50/p65 activity positively modulates innate immunity and inflammation. Nevertheless, expression of these proteins does not implicate activation of the NF-kB pathway. Nuclear factor–kappaB activation has been evaluated in VOL. 98 NO. 3 / SEPTEMBER 2012
Nuclear factor–kappaB (NF-kB) activation pathways. This is a simplified illustration showing the canonical and atypical NF-kB activation pathways (alternative pathway is not considered, because it has not been studied in the context of endometriosis). P ¼ phosphorylation; U ¼ ubiquitination. Gonz alez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
endometrial cells in vitro, which have shown basal NF-kB activity (35, 36). In human endometrium in vivo, NF-kB– DNA binding has been shown to be variable across the menstrual cycle, and higher p65-DNA binding was shown in the proliferative endometrium than in the secretory and menstrual endometrium (34, 37). The increased p65 activity in proliferative endometrium could be modulated by E2 stimulus in the presence of low P concentrations, because 17-b E2 has been shown to activate NF-kB in endometrial epithelial cells (38). This augmentation of p65-DNA binding in proliferative endometrium could modulate increased cell proliferation and decreased apoptosis in endometrial cells during the preovulatory phase of the menstrual cycle, because these are recognized functions consequent to NF-kB transcriptional activity (11, 13, 21, 22). The decrease in NF-kB–p65 activity in secretory endometrium can be explained by the action of high P level in the luteal phase of the menstrual cycle. Progesterone is a known inhibitor of NF-kB (39–42). Progesterone withdrawal during menstruation should theoretically increase NF-kB activation, but a low level of NF-kB–p65 activity was shown in menstrual endometrium compared with proliferative endometrium (34). This could be explained in part by the presence of low E2 levels in this phase of the cycle. Other studies have suggested increased endometrial NF-kB activation during menstruation, but they did not analyze NF-kB– or p65–DNA binding (31, 32). Cyclic or steroidal regulation of NF-kB activity is plausible in 521
VIEWS AND REVIEWS endometrial cells and many studies have evaluated NF-kB– steroidal receptor interactions in other cell types, but NF-kB activity is also modulated by nonsteroidal stimuli and complex cell-specific interactions that make it difficult to interpret the in vivo pattern of NF-kB activation (15, 16, 40–44).
NF-kB ACTIVATION IN HUMAN ENDOMETRIUM FROM ENDOMETRIOSIS PATIENTS The endometrium of patients with endometriosis has been characterized as molecularly different from the normal endometrium of healthy women (1, 45–47). In this line, the endometrium of endometriosis patients showed a different cyclic response concerning p65–DNA binding activity, as compared with the endometrium of healthy women. Further, endometriosis endometrium showed higher p65– DNA binding in proliferative and secretory endometrium than in menstrual endometrium. The absence of reduced NF-kB–p65 activity in secretory endometrium (as observed in physiologic conditions) is concurrent with P resistance in the endometrium of endometriosis patients (34). In consequence, NF-kB–altered activation in the endometrium of endometriosis patients, characterized by a more constant pattern of activation across the menstrual cycle than in the normal endometrium, may explain some of the endometrial dysfunctions associated with endometriosis, mostly related to an increased inflammatory status, proliferative activity, and resistance to apoptosis of endometrial cells (47–49). If this is a primary condition of the endometrium at the origin of the disease or an effect of endometriosis has not been elucidated as yet. On the other hand, in vitro studies have illustrated differences between eutopic endometrial cells and ectopic endometrial cells, showing higher induced and basal NF-kB activity in ectopic than in eutopic endometrial cells (38, 50, 51), which could be participating in endometriotic lesion survival and lead to a proinflammatory status.
NF-kB–MODULATED INFLAMMATION IN ENDOMETRIOSIS Evidence on NF-kB–modulated inflammation in endometriosis has been deduced by studying peritoneal endometriotic implants and peritoneal fluid in vivo, endometriotic epithelial and stromal cells from ovarian endometriotic cysts in vitro, endometriotic cell line cultures, and animal endometriosis models (39, 50, 52–60). Red peritoneal endometriotic implants are characteristically more inflammatory than black endometriotic lesions (10, 61–65). Nuclear factor–kappaB– DNA and p65–DNA binding, macrophage infiltration, IL-1 receptor 1 (IL-1R1) and intercellular adhesion molecule 1 (ICAM-1) expressions were shown to be higher in red endometriotic lesions than in black endometriotic lesions (10, 63, 66). The increased basal NF-kB activation and inflammatory response in red lesions can be explained by the elevated macrophage infiltration and high IL-1R1 expression in these lesions (10, 13). Macrophages in the peritoneal fluid of 522
women with endometriosis showed increased p65 nuclear localization than peritoneal macrophages from control women (12). Stimulation of endometriotic cells in culture with IL-1b and TNF-a positively regulates the expression of multiple proinflammatory cytokines by activating NF-kB. Table 1 shows NF-kB–modulated genes (proteins) that have been shown to participate in endometriosis inflammatory reaction. Using NF-kB inhibitors in endometrial and endometriotic cell cultures and in endometriosis animal models has resulted in a reduction of the expression of most of the NF-kB–regulated cytokines mentioned in Table 1 (11, 31, 39, 50, 53, 56, 57, 59, 60, 69, 74, 77–81). Nuclear factor–kappaB activation stimulates the synthesis of its own regulatory protein IkB, inhibiting the pathway. But stimulating the pathway also arouses NF-kB–mediated synthesis of proinflammatory cytokines such as RANTES (regulated on activation, normal T cell expressed and secreted), ICAM-1, IL-1, or TNF-a, thereby bringing positive NF-kB feedback and enhancing macrophage recruitment and the inflammatory response in endometriosis (13, 22, 23, 82, 83).
NF-kB–REGULATED CELL PROLIFERATION AND APOPTOSIS IN ENDOMETRIOSIS Activation of p50/p65 NF-kB dimers through the canonical or atypical NF-kB pathways modulates the transcription of many genes that stimulate cell proliferation and inhibit apoptosis (17–22). Animal models of endometriosis and in vitro studies in endometriotic cells have shown NFkB–dependent modulation of genes that augment cell proliferation and reduce apoptosis. These genes (and respective proteins) are shown in Table 1. Inhibition of NFkB in a mouse model of endometriosis induced with human menstrual endometrium has resulted in a reduction of endometriotic lesion development by decreasing cell proliferation and increasing apoptosis of endometriotic epithelial and stromal cells (11). A study in p50 knockout mice showed a reduction in endometriotic implants size relative to endometriotic implants in wild type mice (84). Other animal studies have shown diminished endometriotic lesion development and cell proliferation in response to NFkB inhibition (71, 80, 85). In endometriotic cells in culture, inhibition of NF-kB reduced cell proliferation and stimulated apoptosis by down-regulating IL-8, macrophagemigration inhibitory factor, survivin, B-cell lymphoma/leukemia 2 (Bcl-2) and Bcl-XL (antiapoptotic proteins at the mitochondrial level), and by activating caspase 3, caspase 8, and caspase 9 (54, 58, 59, 69, 74, 75). In summary, NF-kB is a main regulator of cell survival in endometriotic lesions, promoting endometriotic cell proliferation and inhibiting apoptosis.
INVOLVEMENT OF PERITONEAL IRON OVERLOAD IN ENDOMETRIOSIS Although iron is an essential metal for almost all living organisms, excess iron accumulation within tissues and cells can result in toxicity and is associated with the pathogenesis of a variety of diseases, such as thalassemia, VOL. 98 NO. 3 / SEPTEMBER 2012
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TABLE 1 NF-kB–up-regulated genes (proteins) involved in inflammation and growth in endometriotic cells. Gene (protein)
Cell type
Model
CCL5 (RANTES)
ESC EEC ESC ESC EEC ESC EEC EEC EEC EEC ESC ESC EEC ESC EEC ESC ESC EEC ESC ESC ESC ESC EEC ESC EEC EEC ESC ESC EEC ESC EEC EEC
In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vivo, in vitro In vitro In vitro In vitro In vitro In vivo, in vitro In vitro In vitro In vivo In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vivo, in vitro In vivo, in vitro In vitro In vitro In vitro In vitro In vitro
MIF (MIF) IL8 (IL8) IL6 (IL6) TNF (TNF-a) CSF2 (GM-CSF) CD54 (ICAM-1) VCAM1 (VCAM-1) CCL2 (MCP-1) PTGS2 (COX-2) F3 (TF-1) NGFB (NGF) CD44 (CD44) MMP9 (MMP-9) BIRC5 (Survivin)a BCL2 (Bcl-2) BCL2L1 (Bcl-XL) b
CASP3 (Caspase-3)
CASP8 (Caspase-8)b b
CASP9 (Caspase-9) XIAP (XIAP)
Function Chemotactic protein, inflammation Inflammation, growth factor Inflammatory chemokine, growth factor Inflammatory cytokine Inflammation Growth factor Cell adhesion molecule Cell adhesion molecule Chemotactic protein, inflammation Stress response gene, proinflammatory Acute phase protein Nerve growth factor Cell adhesion molecule Collagenase, invasion Antiapoptotic Prosurvival factor Prosurvival factor Proapoptotic protein Proapoptotic protein Proapoptotic protein Inhibitor of apoptosis
References (52, 60) (57) (59) (53, 54, 56, 58, 67, 68, 69) (57) (55, 58, 68) (57) (57) (57) (11, 57) (68) (68) (57) (68) (70, 71) (71, 72) (71) (71) (73) (73) (73) (74, 75) (75) (74, 75) (75, 76) (11, 76) (11, 74) (74) (76) (74) (76) (76)
Note: RANTES ¼ regulated on activation, normal T cell expressed and secreted; MIF ¼ macrophage migration inhibitory factor; IL ¼ interleukin; TNF ¼ tumor necrosis factor; GM-CSF ¼ granulocyte macrophage–colony-stimulating factor; ICAM ¼ intercellular adhesion molecule; VCAM ¼ vascular cellular adhesion molecule; MCP ¼ monocyte chemoattractant protein; COX ¼ cyclooxygenase; TF ¼ tissue factor; NGF ¼ nerve growth factor; MMP ¼ matrix metalloproteinase; Bcl ¼ B-cell lymphoma/leukemia; XIAP ¼ X-linked inhibitor of apoptosis protein; ESC ¼ endometriotic stromal cells; EEC ¼ endometriotic epithelial cells. a Evidence point to indirect BIRC5 up-regulation by NF-kB. b CASP3, CASP8, and CASP9 are not NF-kB target genes, but NF-kB inhibition in these studies has resulted in increased caspase-3, -8 and -9 activity or expression, putting into evidence the downregulatory action of NF-kB on apoptosis. Gonzalez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
hemochromatosis, HIV, and neurodegenerative disorders (86). Similarly, iron overload has been involved in endometriosis development (87) by affecting endometrial tissue adhesion, endometriotic lesion proliferation, angiogenesis, and endometriosis-associated subfertility (14, 88). Peritoneal iron overload in case of endometriosis has been highlighted in the different compartments of the peritoneal cavity: peritoneal fluid (89–96), ectopic endometrial tissue (97–102), and peritoneum adjacent to lesions (93). Mounting evidence suggests that iron metabolism by peritoneal macrophages is enhanced in the case of endometriosis. This is supported by the observation that endometriosis is characterized by the presence of macrophages heavily laden with hemosiderin (103, 104) or ferritin (95) inside the pelvic cavity. Most cells protect themselves from iron toxicity by expressing inducible heme oxygenase 1 and scavenger proteins such as haptoglobin and hemopexin, binding hemoglobin and heme, respectively. However, the increased iron load, observed in all compartments of the peritoneal cavity in endometriosis patients compared with controls, strongly VOL. 98 NO. 3 / SEPTEMBER 2012
suggests that iron homeostasis in the peritoneal cavity may be disrupted in these patients (14, 88). Iron overload in the pelvic cavity of endometriosis patients has been postulated as a causal contributing factor and also as a consequence of endometriosis (14, 88, 92, 93, 95).
ORIGIN OF IRON IN THE PELVIC CAVITY IN CASE OF ENDOMETRIOSIS Pelvic iron overload may originate from lysis of erythrocytes (105, 106) transported through the fallopian tubes into the peritoneal cavity by retrograde menstruation. This reflux is a common physiological event in menstruating women (107). Why then does iron accumulate inside the pelvic cavity of some women but not others? One hypothesis is that in some patients peritoneal protective mechanisms might be overwhelmed by menstrual reflux, either because of its abundance, or because of defective scavenging systems (4, 88, 92). In endometriosis patients, retrograde menstruation may be increased by certain anatomical dispositions often 523
VIEWS AND REVIEWS encountered in these patients (108, 109) or heavier menstrual periods than in controls (110–113). Moreover, processes other than menstrual reflux, such as lesion bleeding, may contribute to the accumulation of erythrocytes in peritoneal fluid. Indeed, increased concentrations of erythrocytes have been reported in the peritoneal cavity of women with endometriosis (107, 114).
IRON METABOLISM IN THE PELVIC CAVITY IN CASE OF ENDOMETRIOSIS Studies with experimental models yielded further information on peritoneal iron metabolism in the case of endometriosis. Interpretation of these findings in the light of data on erythrocyte metabolism led to the development of the hypothetical model shown in Figure 2. Within the pelvic cavity, macrophages play an important role in the degradation of erythrocytes and iron recycling. Macrophages phagocytose senescent erythrocytes or endocytose the hemoglobin–haptoglobin complex formed after hemoglobin release by erythrocyte lysis (115). Metabolism of hemoglobin and heme by heme oxygenase releases iron, which is then incorporated into ferritin in macrophages or returned to the iron transporter Tf via peritoneal fluid. Tf may then be assimilated by ectopic endometrial cells. Iron
FIGURE 2
Origin of iron overload in the pelvic cavity of endometriosis patients. Erythrocytes are carried into the pelvic cavity by retrograde menstruation and hemorrhaging foci of ectopic endometrium. A proportion of them are phagocytosed by peritoneal macrophages. Metabolism of heme by heme oxygenase (HO) releases iron. Macrophages store some iron in the form of ferritin or hemosiderin and release some that binds to transferrin (Tf). Macrophages are also able to release ferritin into the peritoneal fluid, whereas lysis of erythrocytes releases hemoglobin (Hb) into the peritoneal fluid. Increased pelvic iron concentrations result from Tf, ferritin, and Hb accumulation in the peritoneal fluid. Tf and Hb may be assimilated by ectopic endometrial cells, resulting in the formation of iron deposits (ferritin or hemosiderin) inside lesions. Gonzalez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
524
is sequestrated within tissue and bound to proteins such as ferritin in a soluble, nontoxic, and bioavailable form. Conglomerates consisting of hemosiderin—another iron storage form—are found in conditions of iron overload and are usually associated with toxic pathological states in humans. Iron conglomerates also have been observed in endometriotic lesions in women (93, 97, 98) and in a murine endometriosis model (105, 106). Furthermore, macrophages are able to release ferritin (115). Iron released by macrophages in the form of ferritin or Tf, or by erythrocyte lysis (hemoglobin), results in increased peritoneal fluid iron concentrations in endometriosis patients (93). Metabolization of heme has also been found to occur within endometrial implants. Indeed, active red endometrial lesions strongly express heme oxygenase, the enzyme catalyzing degradation of the heme moiety of hemoglobin into iron, carbon monoxide, and biliverdin (92, 116). Iron is then incorporated into ferritin or hemosiderin in conditions of iron overload. The presence of hemosiderin in ectopic endometrial tissue and macrophages, usually associated with toxic pathological states in humans, strongly suggests that existing peritoneal protective mechanisms might be overwhelmed in case of endometriosis.
IRON, MACROPHAGES, OXIDATIVE STRESS, AND NF-kB IN ENDOMETRIOSIS Peritoneal macrophages are known to play an important role in the initiation, maintenance, and progression of endometriotic lesions (52, 117). They may exhibit differences in phenotype, as illustrated by higher expression of estrogen receptors a and b, differentiation markers (CD68, NCLMACRO, and HAM56), and inflammatory cytokines (IL-1b, TNF-a, and IL-6) (118). They have been found to be increased in number and more activated in case of endometriosis, releasing various products such as cytokines and growth and angiogenic factors (8, 119, 120). In fact, activation of macrophages is an essential defense mechanism (acute inflammation), but in pathological conditions such as endometriosis, their activation may become exacerbated and inflammation chronic (121). Physiological macrophage functions include phagocytosis, iron metabolism, antimicrobial properties, and TNFmediated cytotoxicity. Peritoneal macrophages are able to remove erythrocytes, damaged tissue fragments, and in all likelihood, endometrial cells from the abdominal cavity. At nontoxic concentrations, iron promotes the physiological functions of macrophages, but iron overload is also known to impair macrophage phagocytosis, respiratory burst, and cytokine expression and is a known inducer of the NF-kB pathway in hepatic macrophages, provoking chronic tissue damage and inflammation (13, 122–124). Lousse et al. recently showed iron storage levels to be higher in peritoneal macrophages of endometriosis patients than controls (95). Cellular iron storage within ferritin limits the capacity of iron to generate free radicals (125). However, continued delivery of iron to macrophages can overwhelm the capacity of ferritin to store and sequester the metal, causing oxidative injury to cells. Indeed, iron can act as VOL. 98 NO. 3 / SEPTEMBER 2012
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FIGURE 3
Iron-mediated NF-kB activation theory. Fe ¼ iron; ROS ¼ reactive oxygen species; p65/p50 ¼ NF-kB dimer; IkB ¼ NF-kB inhibitory protein; IKK ¼ IkB kinase complex; TNF ¼ tumor necrosis factor; IL ¼ interleukin; ICAM ¼ intercellular adhesion molecule; MCP ¼ monocyte chemoattractant protein; RANTES ¼ regulated on activation, normal T cell expressed and secreted; GM-CSF ¼ granulocyte macrophage–colony-stimulating factor; COX ¼ cyclooxygenase; VEGF ¼ vascular endothelial growth factor; MIF ¼ macrophage migration inhibitory factor; MMP ¼ matrix metalloproteinase; Bcl ¼ B-cell lymphoma/leukemia; XIAP ¼ X-linked inhibitor of apoptosis protein. Gonzalez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
a catalyst in the Fenton reaction (Fe2þ þ H2O2 / Fe3þ þ OH þ OH ) to potentiate oxygen and nitrogen toxicity by generation of a wide range of free radical species, including hydroxyl radicals, OH , or the peroxynitrite anion (ONOO) produced by a reaction between nitric oxide (NO) and the superoxide anion (O2). Hydroxyl radicals are among the most reactive free radical species known and have the ability to react with a wide range of cellular constituents, including amino acid residues and purine and pyrimidine bases of DNA, as well as attack membrane lipids, to initiate a free radical chain reaction known as lipid peroxidation. It is clear that reactive oxygen species (ROS) are generated within the cell in the course of normal cellular mechanisms, and those cells are usually adequately equipped with cytoprotective enzymes and antioxidants to combat their toxicity. However, when the balance between ROS production and antioxidant defense is disrupted, marginally higher levels of ROS are generated and oxidative stress may occur, leading to harmful effects. Oxidative stress has been proposed as a potential factor associated with endometriosis pathophysiology (4, 126–128). Overproduction of ROS not only induces cellular damage, but may also impair cellular function by altering protein activity and gene expression (129). Indeed, ROS play a key role in the regulation of redox-sensitive transcription factors like NF-kB (129), which has been implicated in endometriosis development (9–11, 13). Considering all these evidences, we hypothesize iron overload in the pelvic cavity of endometriosis patients as an
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initial factor at the origin of the disease. Iron overload–mediated oxidative stress may activate NF-kB in peritoneal macrophages, expressing and secreting proinflammatory, growth, and angiogenic factors, such as IL1, IL6, IL8, TNF-a, cyclooxygenase 2, and vascular endothelial growth factor (2, 12, 49, 120, 122, 130, 131), also activating NF-kB in endometriotic cells, increasing NF-kB target gene transcription in these cells. This vicious circle consequently promotes and perpetuates cell survival, growth, and inflammation in endometriosis (Fig. 3). In conclusion, the pleiotropic factor NF-kB exerts important cell functions in endometrial physiology and it has been shown to be dysregulated in the endometrium from endometriosis patients and in endometriotic lesions. The main cell processes that NF-kB regulates, contributing to endometriosis development, are inflammation, cell proliferation, and inhibition of apoptosis. Iron overload in the pelvic cavity of endometriosis patients is very probably an important facilitator or inductor of chronic NF-kB activation, enhancing the NF-kB– mediated inflammatory reaction and endometriotic cell survival and growth.
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Instituto de Investigaciones Materno Infantil, Departamento de Obstetricia y Ginecología, Hospital Clínico San n, Facultad de Medicina, Universidad de Chile, Santiago, Chile; and b Department of Gynecology, Universite Borja-Arriara Catholique de Louvain, Brussels, Belgium
Objective: To update, analyze, and summarize the literature concerning nuclear factor–kappaB (NF-kB) participation in endometriosis pathophysiology. Design: Review. Result(s): Nuclear factor–kappaB is physiologically activated in the human endometrium, showing variable activity. A cyclic p65DNA binding pattern was shown in the endometrium of healthy women. This cyclic pattern was altered in the endometrium of patients with endometriosis. Nuclear factor–kappaB is basally activated in peritoneal endometriotic lesions, showing higher p65 activity in red endometriotic lesions than in black lesions. In vivo and in vitro studies show up-regulation of inflammation and cell proliferation and down-regulation of apoptosis by NF-kB activity. Iron overload has been shown in the pelvic cavity of endometriosis patients, and iron overload and oxidative stress activate NF-kB in macrophages, which have been shown to participate in the endometriosis-associated inflammatory reaction. Conclusion(s): Nuclear factor–kappaB activation dysregulation in the endometrium of endometriosis patients may explain some endometrial biological alterations associated with endometriosis. The scientific evidence strongly suggests that NF-kB activity in endometriotic cells stimulates inflammation and cell proliferation and inhibits apoptosis, favoring the development and maintenance of endometriosis. Iron overload in the pelvic cavity of endometriosis patients could be a main factor enhancing oxidative stress and activating NF-kB in a chronic manner, contributing to endometriosis establishment and growth. Use your smartphone (Fertil SterilÒ 2012;98:520–8. Ó2012 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Apoptosis, cell proliferation, endometriosis, endometrium, inflammation, iron, and connect to the macrophages, nuclear factor–kappaB Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/gonzalez-ramosr-nuclear-factor-kappab-inflammation-cell-survival-inendometriosis/
T
he survival of endometrial cells on ectopic locations, named endometriosis, is a complex pathophysiological phenomenon. Numerous proinflammatory cytokines and growth factors have been involved in the development and maintenance of this disease (1–8). Most of the main proteins that have been implicated in
an endometriosis-associated proinflammatory environment are up-regulated by the transcription factor nuclear factor–kappaB (NF-kB). The proinflammatory peptides implied in this local event in the pelvic cavity of endometriosis patients contribute to stimulate cell proliferation and inhibit apoptosis of endometriotic cells,
Received May 18, 2012; revised June 5, 2012; accepted June 8, 2012; published online July 6, 2012. R.G.-R. has nothing to disclose. S.D. has nothing to disclose. L.D. has nothing to disclose. lez-Ramos, M.D., Ph.D., IDIMI, Facultad de Medicina Centro, UniverReprint requests: Reinaldo Gonza n, Av. Sta. Rosa 1234, 2 piso, Santiago, Chile sidad de Chile, Hospital Clínico San Borja Arriara (E-mail: [email protected]). Fertility and Sterility® Vol. 98, No. 3, September 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.06.021 520
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facilitating endometriotic cell survival (9–14). Nuclear factor–kappaB has been shown to be a key protein favoring cell proliferation and inhibiting apoptosis in various cell types, including endometrial and endometriotic cells (11, 13, 15–22). This review is focused on NF-kB– controlled gene transcription, NF-kB signaling, and interactions involved in endometriosis pathophysiology and particularly in endometriosis-related inflammatory process and endometriotic cell survival. In addition, because iron overload in the pelvic cavity of endometriosis patients has been identified VOL. 98 NO. 3 / SEPTEMBER 2012
Fertility and Sterility® as a possible mechanism at the origin of endometriosis, and evidence points to iron-mediated oxidative stress as an NFkB activating factor, the literature concerning iron overload in endometriosis and iron overload–dependent NF-kB activation will be examined as a possible factor contributing to the pathogenesis of endometriosis.
FIGURE 1
NF-kB The transcription factor NF-kB is a family of peptides formed by five subunits: RelA or p65, RelB, c-Rel, p50/p105, and p52/ p100. These subunits bind forming different NF-kB dimers that remain inactive by their union to the NF-kB inhibitory protein IkB. This protein prevents NF-kB–DNA interaction, and thus NF-kB target genes transcription is blocked (23–29). The canonical NF-kB pathway is activated by proinflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1b and by lipopolysaccharide (LPS) derived from bacteria that stimulate the I-kappaB kinase complex (IKK), phosphorylating IkB. IkB phosphorylation and subsequent ubiquitination allow its degradation by the proteasome. As a result, NF-kB free dimers are able to bind to DNA, initiating NF-kB target genes transcription. This pathway activates mainly p50/p65 NF-kB dimers that have been involved in the transcription of genes that regulate innate immunity, inflammation, and cell survival. Atypical pathways activating also p50/p65 NF-kB dimers via tyrosine kinase (TyrK) or casein kinase-II (CK2) are triggered by stimuli such as hypoxia, oxidative stress (which can be caused or favored by iron overload), and ultraviolet radiations (Fig. 1). Other important NF-kB dimers regulating gene transcription are p50/p50 dimers, which lack the transcriptional activation domain but bind to DNA because they have the Rel homology domain like all NF-kB subunits. Thus p50/p50 dimers are transcriptionally inactive, and consequently they have been linked to inflammation ceasing. Nuclear factor–kappaB p52/RelB dimers are activated by alternative pathways and they have been associated with lymphoid organogenesis, B-cell maturation, and humoral immunity (16, 29, 30). Research on NF-kB and endometriosis has been carried out mainly considering the canonical or atypical activation pathways, which lead to the activation of p50/p65 dimers, and this paper relates consequently.
PHYSIOLOGIC ACTIVATION OF NF-kB IN THE HUMAN ENDOMETRIUM Nuclear factor–kappaB p50 and p65 subunits, IkB and IKK are expressed in human endometrial stromal and epithelial cells (31–33). The expression of p65 is higher in the endometrial epithelium than in the stroma (34), which could mean that the canonical and/or atypical NF-kB pathways are physiologically more important in the endometrial epithelium than in the stroma. This seems logical, because the endometrial epithelium is the first defensive barrier in case of infections, and NF-kB p50/p65 activity positively modulates innate immunity and inflammation. Nevertheless, expression of these proteins does not implicate activation of the NF-kB pathway. Nuclear factor–kappaB activation has been evaluated in VOL. 98 NO. 3 / SEPTEMBER 2012
Nuclear factor–kappaB (NF-kB) activation pathways. This is a simplified illustration showing the canonical and atypical NF-kB activation pathways (alternative pathway is not considered, because it has not been studied in the context of endometriosis). P ¼ phosphorylation; U ¼ ubiquitination. Gonz alez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
endometrial cells in vitro, which have shown basal NF-kB activity (35, 36). In human endometrium in vivo, NF-kB– DNA binding has been shown to be variable across the menstrual cycle, and higher p65-DNA binding was shown in the proliferative endometrium than in the secretory and menstrual endometrium (34, 37). The increased p65 activity in proliferative endometrium could be modulated by E2 stimulus in the presence of low P concentrations, because 17-b E2 has been shown to activate NF-kB in endometrial epithelial cells (38). This augmentation of p65-DNA binding in proliferative endometrium could modulate increased cell proliferation and decreased apoptosis in endometrial cells during the preovulatory phase of the menstrual cycle, because these are recognized functions consequent to NF-kB transcriptional activity (11, 13, 21, 22). The decrease in NF-kB–p65 activity in secretory endometrium can be explained by the action of high P level in the luteal phase of the menstrual cycle. Progesterone is a known inhibitor of NF-kB (39–42). Progesterone withdrawal during menstruation should theoretically increase NF-kB activation, but a low level of NF-kB–p65 activity was shown in menstrual endometrium compared with proliferative endometrium (34). This could be explained in part by the presence of low E2 levels in this phase of the cycle. Other studies have suggested increased endometrial NF-kB activation during menstruation, but they did not analyze NF-kB– or p65–DNA binding (31, 32). Cyclic or steroidal regulation of NF-kB activity is plausible in 521
VIEWS AND REVIEWS endometrial cells and many studies have evaluated NF-kB– steroidal receptor interactions in other cell types, but NF-kB activity is also modulated by nonsteroidal stimuli and complex cell-specific interactions that make it difficult to interpret the in vivo pattern of NF-kB activation (15, 16, 40–44).
NF-kB ACTIVATION IN HUMAN ENDOMETRIUM FROM ENDOMETRIOSIS PATIENTS The endometrium of patients with endometriosis has been characterized as molecularly different from the normal endometrium of healthy women (1, 45–47). In this line, the endometrium of endometriosis patients showed a different cyclic response concerning p65–DNA binding activity, as compared with the endometrium of healthy women. Further, endometriosis endometrium showed higher p65– DNA binding in proliferative and secretory endometrium than in menstrual endometrium. The absence of reduced NF-kB–p65 activity in secretory endometrium (as observed in physiologic conditions) is concurrent with P resistance in the endometrium of endometriosis patients (34). In consequence, NF-kB–altered activation in the endometrium of endometriosis patients, characterized by a more constant pattern of activation across the menstrual cycle than in the normal endometrium, may explain some of the endometrial dysfunctions associated with endometriosis, mostly related to an increased inflammatory status, proliferative activity, and resistance to apoptosis of endometrial cells (47–49). If this is a primary condition of the endometrium at the origin of the disease or an effect of endometriosis has not been elucidated as yet. On the other hand, in vitro studies have illustrated differences between eutopic endometrial cells and ectopic endometrial cells, showing higher induced and basal NF-kB activity in ectopic than in eutopic endometrial cells (38, 50, 51), which could be participating in endometriotic lesion survival and lead to a proinflammatory status.
NF-kB–MODULATED INFLAMMATION IN ENDOMETRIOSIS Evidence on NF-kB–modulated inflammation in endometriosis has been deduced by studying peritoneal endometriotic implants and peritoneal fluid in vivo, endometriotic epithelial and stromal cells from ovarian endometriotic cysts in vitro, endometriotic cell line cultures, and animal endometriosis models (39, 50, 52–60). Red peritoneal endometriotic implants are characteristically more inflammatory than black endometriotic lesions (10, 61–65). Nuclear factor–kappaB– DNA and p65–DNA binding, macrophage infiltration, IL-1 receptor 1 (IL-1R1) and intercellular adhesion molecule 1 (ICAM-1) expressions were shown to be higher in red endometriotic lesions than in black endometriotic lesions (10, 63, 66). The increased basal NF-kB activation and inflammatory response in red lesions can be explained by the elevated macrophage infiltration and high IL-1R1 expression in these lesions (10, 13). Macrophages in the peritoneal fluid of 522
women with endometriosis showed increased p65 nuclear localization than peritoneal macrophages from control women (12). Stimulation of endometriotic cells in culture with IL-1b and TNF-a positively regulates the expression of multiple proinflammatory cytokines by activating NF-kB. Table 1 shows NF-kB–modulated genes (proteins) that have been shown to participate in endometriosis inflammatory reaction. Using NF-kB inhibitors in endometrial and endometriotic cell cultures and in endometriosis animal models has resulted in a reduction of the expression of most of the NF-kB–regulated cytokines mentioned in Table 1 (11, 31, 39, 50, 53, 56, 57, 59, 60, 69, 74, 77–81). Nuclear factor–kappaB activation stimulates the synthesis of its own regulatory protein IkB, inhibiting the pathway. But stimulating the pathway also arouses NF-kB–mediated synthesis of proinflammatory cytokines such as RANTES (regulated on activation, normal T cell expressed and secreted), ICAM-1, IL-1, or TNF-a, thereby bringing positive NF-kB feedback and enhancing macrophage recruitment and the inflammatory response in endometriosis (13, 22, 23, 82, 83).
NF-kB–REGULATED CELL PROLIFERATION AND APOPTOSIS IN ENDOMETRIOSIS Activation of p50/p65 NF-kB dimers through the canonical or atypical NF-kB pathways modulates the transcription of many genes that stimulate cell proliferation and inhibit apoptosis (17–22). Animal models of endometriosis and in vitro studies in endometriotic cells have shown NFkB–dependent modulation of genes that augment cell proliferation and reduce apoptosis. These genes (and respective proteins) are shown in Table 1. Inhibition of NFkB in a mouse model of endometriosis induced with human menstrual endometrium has resulted in a reduction of endometriotic lesion development by decreasing cell proliferation and increasing apoptosis of endometriotic epithelial and stromal cells (11). A study in p50 knockout mice showed a reduction in endometriotic implants size relative to endometriotic implants in wild type mice (84). Other animal studies have shown diminished endometriotic lesion development and cell proliferation in response to NFkB inhibition (71, 80, 85). In endometriotic cells in culture, inhibition of NF-kB reduced cell proliferation and stimulated apoptosis by down-regulating IL-8, macrophagemigration inhibitory factor, survivin, B-cell lymphoma/leukemia 2 (Bcl-2) and Bcl-XL (antiapoptotic proteins at the mitochondrial level), and by activating caspase 3, caspase 8, and caspase 9 (54, 58, 59, 69, 74, 75). In summary, NF-kB is a main regulator of cell survival in endometriotic lesions, promoting endometriotic cell proliferation and inhibiting apoptosis.
INVOLVEMENT OF PERITONEAL IRON OVERLOAD IN ENDOMETRIOSIS Although iron is an essential metal for almost all living organisms, excess iron accumulation within tissues and cells can result in toxicity and is associated with the pathogenesis of a variety of diseases, such as thalassemia, VOL. 98 NO. 3 / SEPTEMBER 2012
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TABLE 1 NF-kB–up-regulated genes (proteins) involved in inflammation and growth in endometriotic cells. Gene (protein)
Cell type
Model
CCL5 (RANTES)
ESC EEC ESC ESC EEC ESC EEC EEC EEC EEC ESC ESC EEC ESC EEC ESC ESC EEC ESC ESC ESC ESC EEC ESC EEC EEC ESC ESC EEC ESC EEC EEC
In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vivo, in vitro In vitro In vitro In vitro In vitro In vivo, in vitro In vitro In vitro In vivo In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vivo, in vitro In vivo, in vitro In vitro In vitro In vitro In vitro In vitro
MIF (MIF) IL8 (IL8) IL6 (IL6) TNF (TNF-a) CSF2 (GM-CSF) CD54 (ICAM-1) VCAM1 (VCAM-1) CCL2 (MCP-1) PTGS2 (COX-2) F3 (TF-1) NGFB (NGF) CD44 (CD44) MMP9 (MMP-9) BIRC5 (Survivin)a BCL2 (Bcl-2) BCL2L1 (Bcl-XL) b
CASP3 (Caspase-3)
CASP8 (Caspase-8)b b
CASP9 (Caspase-9) XIAP (XIAP)
Function Chemotactic protein, inflammation Inflammation, growth factor Inflammatory chemokine, growth factor Inflammatory cytokine Inflammation Growth factor Cell adhesion molecule Cell adhesion molecule Chemotactic protein, inflammation Stress response gene, proinflammatory Acute phase protein Nerve growth factor Cell adhesion molecule Collagenase, invasion Antiapoptotic Prosurvival factor Prosurvival factor Proapoptotic protein Proapoptotic protein Proapoptotic protein Inhibitor of apoptosis
References (52, 60) (57) (59) (53, 54, 56, 58, 67, 68, 69) (57) (55, 58, 68) (57) (57) (57) (11, 57) (68) (68) (57) (68) (70, 71) (71, 72) (71) (71) (73) (73) (73) (74, 75) (75) (74, 75) (75, 76) (11, 76) (11, 74) (74) (76) (74) (76) (76)
Note: RANTES ¼ regulated on activation, normal T cell expressed and secreted; MIF ¼ macrophage migration inhibitory factor; IL ¼ interleukin; TNF ¼ tumor necrosis factor; GM-CSF ¼ granulocyte macrophage–colony-stimulating factor; ICAM ¼ intercellular adhesion molecule; VCAM ¼ vascular cellular adhesion molecule; MCP ¼ monocyte chemoattractant protein; COX ¼ cyclooxygenase; TF ¼ tissue factor; NGF ¼ nerve growth factor; MMP ¼ matrix metalloproteinase; Bcl ¼ B-cell lymphoma/leukemia; XIAP ¼ X-linked inhibitor of apoptosis protein; ESC ¼ endometriotic stromal cells; EEC ¼ endometriotic epithelial cells. a Evidence point to indirect BIRC5 up-regulation by NF-kB. b CASP3, CASP8, and CASP9 are not NF-kB target genes, but NF-kB inhibition in these studies has resulted in increased caspase-3, -8 and -9 activity or expression, putting into evidence the downregulatory action of NF-kB on apoptosis. Gonzalez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
hemochromatosis, HIV, and neurodegenerative disorders (86). Similarly, iron overload has been involved in endometriosis development (87) by affecting endometrial tissue adhesion, endometriotic lesion proliferation, angiogenesis, and endometriosis-associated subfertility (14, 88). Peritoneal iron overload in case of endometriosis has been highlighted in the different compartments of the peritoneal cavity: peritoneal fluid (89–96), ectopic endometrial tissue (97–102), and peritoneum adjacent to lesions (93). Mounting evidence suggests that iron metabolism by peritoneal macrophages is enhanced in the case of endometriosis. This is supported by the observation that endometriosis is characterized by the presence of macrophages heavily laden with hemosiderin (103, 104) or ferritin (95) inside the pelvic cavity. Most cells protect themselves from iron toxicity by expressing inducible heme oxygenase 1 and scavenger proteins such as haptoglobin and hemopexin, binding hemoglobin and heme, respectively. However, the increased iron load, observed in all compartments of the peritoneal cavity in endometriosis patients compared with controls, strongly VOL. 98 NO. 3 / SEPTEMBER 2012
suggests that iron homeostasis in the peritoneal cavity may be disrupted in these patients (14, 88). Iron overload in the pelvic cavity of endometriosis patients has been postulated as a causal contributing factor and also as a consequence of endometriosis (14, 88, 92, 93, 95).
ORIGIN OF IRON IN THE PELVIC CAVITY IN CASE OF ENDOMETRIOSIS Pelvic iron overload may originate from lysis of erythrocytes (105, 106) transported through the fallopian tubes into the peritoneal cavity by retrograde menstruation. This reflux is a common physiological event in menstruating women (107). Why then does iron accumulate inside the pelvic cavity of some women but not others? One hypothesis is that in some patients peritoneal protective mechanisms might be overwhelmed by menstrual reflux, either because of its abundance, or because of defective scavenging systems (4, 88, 92). In endometriosis patients, retrograde menstruation may be increased by certain anatomical dispositions often 523
VIEWS AND REVIEWS encountered in these patients (108, 109) or heavier menstrual periods than in controls (110–113). Moreover, processes other than menstrual reflux, such as lesion bleeding, may contribute to the accumulation of erythrocytes in peritoneal fluid. Indeed, increased concentrations of erythrocytes have been reported in the peritoneal cavity of women with endometriosis (107, 114).
IRON METABOLISM IN THE PELVIC CAVITY IN CASE OF ENDOMETRIOSIS Studies with experimental models yielded further information on peritoneal iron metabolism in the case of endometriosis. Interpretation of these findings in the light of data on erythrocyte metabolism led to the development of the hypothetical model shown in Figure 2. Within the pelvic cavity, macrophages play an important role in the degradation of erythrocytes and iron recycling. Macrophages phagocytose senescent erythrocytes or endocytose the hemoglobin–haptoglobin complex formed after hemoglobin release by erythrocyte lysis (115). Metabolism of hemoglobin and heme by heme oxygenase releases iron, which is then incorporated into ferritin in macrophages or returned to the iron transporter Tf via peritoneal fluid. Tf may then be assimilated by ectopic endometrial cells. Iron
FIGURE 2
Origin of iron overload in the pelvic cavity of endometriosis patients. Erythrocytes are carried into the pelvic cavity by retrograde menstruation and hemorrhaging foci of ectopic endometrium. A proportion of them are phagocytosed by peritoneal macrophages. Metabolism of heme by heme oxygenase (HO) releases iron. Macrophages store some iron in the form of ferritin or hemosiderin and release some that binds to transferrin (Tf). Macrophages are also able to release ferritin into the peritoneal fluid, whereas lysis of erythrocytes releases hemoglobin (Hb) into the peritoneal fluid. Increased pelvic iron concentrations result from Tf, ferritin, and Hb accumulation in the peritoneal fluid. Tf and Hb may be assimilated by ectopic endometrial cells, resulting in the formation of iron deposits (ferritin or hemosiderin) inside lesions. Gonzalez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
524
is sequestrated within tissue and bound to proteins such as ferritin in a soluble, nontoxic, and bioavailable form. Conglomerates consisting of hemosiderin—another iron storage form—are found in conditions of iron overload and are usually associated with toxic pathological states in humans. Iron conglomerates also have been observed in endometriotic lesions in women (93, 97, 98) and in a murine endometriosis model (105, 106). Furthermore, macrophages are able to release ferritin (115). Iron released by macrophages in the form of ferritin or Tf, or by erythrocyte lysis (hemoglobin), results in increased peritoneal fluid iron concentrations in endometriosis patients (93). Metabolization of heme has also been found to occur within endometrial implants. Indeed, active red endometrial lesions strongly express heme oxygenase, the enzyme catalyzing degradation of the heme moiety of hemoglobin into iron, carbon monoxide, and biliverdin (92, 116). Iron is then incorporated into ferritin or hemosiderin in conditions of iron overload. The presence of hemosiderin in ectopic endometrial tissue and macrophages, usually associated with toxic pathological states in humans, strongly suggests that existing peritoneal protective mechanisms might be overwhelmed in case of endometriosis.
IRON, MACROPHAGES, OXIDATIVE STRESS, AND NF-kB IN ENDOMETRIOSIS Peritoneal macrophages are known to play an important role in the initiation, maintenance, and progression of endometriotic lesions (52, 117). They may exhibit differences in phenotype, as illustrated by higher expression of estrogen receptors a and b, differentiation markers (CD68, NCLMACRO, and HAM56), and inflammatory cytokines (IL-1b, TNF-a, and IL-6) (118). They have been found to be increased in number and more activated in case of endometriosis, releasing various products such as cytokines and growth and angiogenic factors (8, 119, 120). In fact, activation of macrophages is an essential defense mechanism (acute inflammation), but in pathological conditions such as endometriosis, their activation may become exacerbated and inflammation chronic (121). Physiological macrophage functions include phagocytosis, iron metabolism, antimicrobial properties, and TNFmediated cytotoxicity. Peritoneal macrophages are able to remove erythrocytes, damaged tissue fragments, and in all likelihood, endometrial cells from the abdominal cavity. At nontoxic concentrations, iron promotes the physiological functions of macrophages, but iron overload is also known to impair macrophage phagocytosis, respiratory burst, and cytokine expression and is a known inducer of the NF-kB pathway in hepatic macrophages, provoking chronic tissue damage and inflammation (13, 122–124). Lousse et al. recently showed iron storage levels to be higher in peritoneal macrophages of endometriosis patients than controls (95). Cellular iron storage within ferritin limits the capacity of iron to generate free radicals (125). However, continued delivery of iron to macrophages can overwhelm the capacity of ferritin to store and sequester the metal, causing oxidative injury to cells. Indeed, iron can act as VOL. 98 NO. 3 / SEPTEMBER 2012
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FIGURE 3
Iron-mediated NF-kB activation theory. Fe ¼ iron; ROS ¼ reactive oxygen species; p65/p50 ¼ NF-kB dimer; IkB ¼ NF-kB inhibitory protein; IKK ¼ IkB kinase complex; TNF ¼ tumor necrosis factor; IL ¼ interleukin; ICAM ¼ intercellular adhesion molecule; MCP ¼ monocyte chemoattractant protein; RANTES ¼ regulated on activation, normal T cell expressed and secreted; GM-CSF ¼ granulocyte macrophage–colony-stimulating factor; COX ¼ cyclooxygenase; VEGF ¼ vascular endothelial growth factor; MIF ¼ macrophage migration inhibitory factor; MMP ¼ matrix metalloproteinase; Bcl ¼ B-cell lymphoma/leukemia; XIAP ¼ X-linked inhibitor of apoptosis protein. Gonzalez-Ramos. NF-kB and endometriosis pathophysiology. Fertil Steril 2012.
a catalyst in the Fenton reaction (Fe2þ þ H2O2 / Fe3þ þ OH þ OH ) to potentiate oxygen and nitrogen toxicity by generation of a wide range of free radical species, including hydroxyl radicals, OH , or the peroxynitrite anion (ONOO) produced by a reaction between nitric oxide (NO) and the superoxide anion (O2). Hydroxyl radicals are among the most reactive free radical species known and have the ability to react with a wide range of cellular constituents, including amino acid residues and purine and pyrimidine bases of DNA, as well as attack membrane lipids, to initiate a free radical chain reaction known as lipid peroxidation. It is clear that reactive oxygen species (ROS) are generated within the cell in the course of normal cellular mechanisms, and those cells are usually adequately equipped with cytoprotective enzymes and antioxidants to combat their toxicity. However, when the balance between ROS production and antioxidant defense is disrupted, marginally higher levels of ROS are generated and oxidative stress may occur, leading to harmful effects. Oxidative stress has been proposed as a potential factor associated with endometriosis pathophysiology (4, 126–128). Overproduction of ROS not only induces cellular damage, but may also impair cellular function by altering protein activity and gene expression (129). Indeed, ROS play a key role in the regulation of redox-sensitive transcription factors like NF-kB (129), which has been implicated in endometriosis development (9–11, 13). Considering all these evidences, we hypothesize iron overload in the pelvic cavity of endometriosis patients as an
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initial factor at the origin of the disease. Iron overload–mediated oxidative stress may activate NF-kB in peritoneal macrophages, expressing and secreting proinflammatory, growth, and angiogenic factors, such as IL1, IL6, IL8, TNF-a, cyclooxygenase 2, and vascular endothelial growth factor (2, 12, 49, 120, 122, 130, 131), also activating NF-kB in endometriotic cells, increasing NF-kB target gene transcription in these cells. This vicious circle consequently promotes and perpetuates cell survival, growth, and inflammation in endometriosis (Fig. 3). In conclusion, the pleiotropic factor NF-kB exerts important cell functions in endometrial physiology and it has been shown to be dysregulated in the endometrium from endometriosis patients and in endometriotic lesions. The main cell processes that NF-kB regulates, contributing to endometriosis development, are inflammation, cell proliferation, and inhibition of apoptosis. Iron overload in the pelvic cavity of endometriosis patients is very probably an important facilitator or inductor of chronic NF-kB activation, enhancing the NF-kB– mediated inflammatory reaction and endometriotic cell survival and growth.
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