EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells.

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Сopper nanoparticles supported on charcoal and betacellulin - Two novel stimulators of ovarian granulosa cell functions and their functional interrelationships.

The present experiments are aimed to examine the effect of copper nanoparticles supported on charcoal (CuNPs/C), growth factor betacellulin (BTC) and their interrelationships in the control of ovarian cell functions. Porcine ovarian granulosa cells were cultured in the presence of CuNPs/C (0, 1, 10 or 100 ng/ml), BTC (100 ng/ml) and the combination of both, CuNPs/C + BTC. Markers of cell proliferation (BrDU incorporation), of the S-phase (PCNA) and G-phase (cyclin B1) of the cell cycle, markers of extrinsic (nuclear DNA fragmentation) and cytoplasmic/mitochondrial apoptosis (bax and caspase 3), and the release of progesterone and estradiol were assessed by BrDU test, TUNEL, quantitative immunocytochemistry and ELISA. Both CuNPs/C and BTC, when added alone, increased the expression of all the markers of cell proliferation, reduced the expression of all apoptosis markers and stimulated progesterone and estradiol release. Moreover, BTC was able to promote the CuNPs/C action on the accumulation of PCNA, cyclin B1, bax and estradiol output. These observations demonstrate the stimulatory action of both CuNPs/C and BTC on ovarian cell functions, as well as the ability of BTC to promote the action of CuNPs/C on ovarian cell functions.

Multi-omic network analysis identified betacellulin as a novel target of omega-3 fatty acid attenuation of western diet-induced nonalcoholic steatohepatitis.

Clinical and preclinical studies established that supplementing diets with ω3 polyunsaturated fatty acids (PUFA) can reduce hepatic dysfunction in nonalcoholic steatohepatitis (NASH) but molecular underpinnings of this action were elusive. Herein, we used multi-omic network analysis that unveiled critical molecular pathways involved in ω3 PUFA effects in a preclinical mouse model of western diet induced NASH. Since NASH is a precursor of liver cancer, we also performed meta-analysis of human liver cancer transcriptomes that uncovered betacellulin as a key EGFR-binding protein upregulated in liver cancer and downregulated by ω3 PUFAs in animals and humans with NASH. We then confirmed that betacellulin acts by promoting proliferation of quiescent hepatic stellate cells, inducing transforming growth factor-ß2 and increasing collagen production. When used in combination with TLR2/4 agonists, betacellulin upregulated integrins in macrophages thereby potentiating inflammation and fibrosis. Taken together, our results suggest that suppression of betacellulin is one of the key mechanisms associated with anti-inflammatory and anti-fibrotic effects of ω3 PUFA on NASH.

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells.

BACKGROUND: The gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined. METHODS: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. RESULTS: The results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells. CONCLUSIONS: BTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalization.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts ionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+ -dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

Experimental and Clinical Evidence Suggests That Treatment with Betacellulin Can Alleviate Th2-Type Cytokine-Mediated Impairment of Skin Barrier Function.

Betacellulin (BTC) is a peptide ligand that belongs to the epidermal growth factor family, the members of which have been implicated in skin morphogenesis, homeostasis, repair, and angiogenesis; however, the role of BTC in the regulation of the skin barrier remains unknown. To examine the role of BTC in skin barrier function, we analyzed atopic dermatitis (AD) transcriptomic data from Gene Expression Omnibus (GEO) datasets, performed BTC immunohistochemistry using human skin tissues, and evaluated the effects of BTC on primary human keratinocytes by real-time PCR, Western blotting, and assay of the transepidermal electrical resistance (TER), a functional parameter to monitor the tight junction barrier. We found that the gene expression of BTC was downregulated in skin lesions from patients with AD, and this downregulated expression recovered following biological treatments. Consistently, the BTC protein levels were downregulated in the lesional skin of AD patients compared with the normal skin of healthy participants, suggesting that the BTC levels in skin might be a biomarker for the diagnosis and therapy of AD. Furthermore, in human keratinocytes, BTC knockdown reduced the levels of skin-derived antimicrobial peptides and skin barrier-related genes, whereas BTC addition enhanced their levels. Importantly, in human skin equivalents, BTC restored the increased tight junction permeability induced by Th2 cytokine IL-4/IL-13 treatment. In addition, specific inhibitors of epidermal growth factor receptor (EGFR) and protein kinase C (PKC) abolished the BTC-mediated improvement in skin barrier-related proteins in keratinocyte monolayers. Collectively, our findings suggest that treatment with BTC might improve the Th2-type cytokine-mediated impairment of skin barrier function through the EGFR/PKC axis and that BTC might be a novel potential biomarker and therapeutic target for the treatment of skin conditions characterized by the overproduction of Th2 cytokines and dysfunctional skin barriers, such as AD.

Betacellulin regulates peripheral nerve regeneration by affecting Schwann cell migration and axon elongation.

BACKGROUND: Growth factors execute essential biological functions and affect various physiological and pathological processes, including peripheral nerve repair and regeneration. Our previous sequencing data showed that the mRNA coding for betacellulin (Btc), an epidermal growth factor protein family member, was up-regulated in rat sciatic nerve segment after nerve injury, implying the potential involvement of Btc during peripheral nerve regeneration. METHODS: Expression of Btc was examined in Schwann cells by immunostaining. The function of Btc in regulating Schwann cells was investigated by transfecting cultured cells with siRNA segment against Btc or treating cells with Btc recombinant protein. The influence of Schwann cell-secreted Btc on neurons was determined using a co-culture assay. The in vivo effects of Btc on Schwann cell migration and axon elongation after rat sciatic nerve injury were further evaluated. RESULTS: Immunostaining images and ELISA outcomes indicated that Btc was present in and secreted by Schwann cells. Transwell migration and wound healing observations showed that transfection with siRNA against Btc impeded Schwann cell migration while application of exogenous Btc advanced Schwann cell migration. Besides the regulating effect on Schwann cell phenotype, Btc secreted by Schwann cells influenced neuron behavior and increased neurite length. In vivo evidence supported the promoting role of Btc in nerve regeneration after both rat sciatic nerve crush injury and transection injury. CONCLUSION: Our findings demonstrate the essential roles of Btc on Schwann cell migration and axon elongation and imply the potential application of Btc as a regenerative strategy for treating peripheral nerve injury.

Formation of ßTC3 and MIN6 Pseudoislets Changes the Expression Pattern of Gpr40, Gpr55, and Gpr119 Receptors and Improves Lysophosphatidylcholines-Potentiated Glucose-Stimulated Insulin Secretion.

The impaired spatial arrangement and connections between cells creating islets of Langerhans as well as altered expression of G protein-coupled receptors (GPCRs) often lead to dysfunction of insulin-secreting pancreatic ß cells and can significantly contribute to the development of diabetes. Differences in glucose-stimulated insulin secretion (GSIS) are noticeable not only in diabetic individuals but also in model pancreatic ß cells, e.g., ßTC3 and MIN6 ß cell lines with impaired and normal insulin secretion, respectively. Now, we compare the ability of GPCR agonists (lysophosphatidylcholines bearing fatty acid chains of different lengths) to potentiate GSIS in ßTC3 and MIN6 ß cell models, cultured as adherent monolayers and in a form of pseudoislets (PIs) with pancreatic MS1 endothelial cells. Our aim was also to investigate differences in expression of the GPCRs responsive to LPCs in these experimental systems. Aggregation of ß cells into islet-like structures greatly enhanced the expression of Gpr40, Gpr55, and Gpr119 receptors. In contrast, the co-culture of ßTC3 cells with endothelial cells converted the GPCR expression pattern closer to the pattern observed in MIN6 cells. Additionally, the efficiencies of various LPC species in ßTC3-MS1 PIs also shifted toward the MIN6 cell model.

Unraveling ERBB network dynamics upon betacellulin signaling in pancreatic ductal adenocarcinoma in mice.

Pancreatic ductal adenocarcinoma (PDAC) will soon belong to the top three cancer killers. The only approved specific PDAC therapy targets the epidermal growth factor receptor (EGFR). Although EGFR is a crucial player in PDAC development, EGFR-based therapy is disappointing. In this study, we evaluated the role of the EGFR ligand betacellulin (BTC) in PDAC. The expression of BTC was investigated in human pancreatic cancer specimen. Then, we generated a BTC knockout mouse model by CRISPR/Cas9 technology and a BTC overexpression model. Both models were crossed with the Ptf1aCre/+ ;KRASG12D/+ (KC) mouse model (B-/- KC or BKC, respectively). In addition, EGFR, ERBB2, and ERBB4 were investigated by the pancreas-specific deletion of each receptor using the Cre-loxP system. Tumor initiation and progression were analyzed in all mouse lines, and the underlying molecular biology of PDAC was investigated at different time points. BTC is expressed in human and murine PDAC. B-/- KC mice showed a decelerated PDAC progression, associated with decreased EGFR activation. BKC mice developed severe PDAC with a poor survival rate. The dramatically increased BTC-mediated tumor burden was EGFR-dependent, but also ERBB4 and ERBB2 were involved in PDAC development or progression, as depletion of EGFR, ERBB2, or ERBB4 significantly improved the survival rate of BTC-mediated PDAC. BTC increases PDAC tumor burden dramatically by enhanced RAS activation. EGFR signaling, ERBB2 signaling, and ERBB4 signaling are involved in accelerated PDAC development mediated by BTC indicating that targeting the whole ERBB family, instead of a single receptor, is a promising strategy for the development of future PDAC therapies.

Betacellulin enhances ovarian cancer cell migration by up-regulating Connexin43 via MEK-ERK signaling.

Epithelial ovarian cancer is the fifth common cause of cancer death in women and the most lethal gynecological malignancies. Our previous studies have shown that up-regulation of Connexin43, a gap-junction subunit crucial for cell-cell communication, enhances ovarian cancer cell migration. Betacellulin is a member of the epidermal growth factor (EGF) family which can bind to multiple EGF family receptors. Overexpression of betacellulin is found in a variety of cancers and is associated with reduced survival. However, the specific roles and molecular mechanisms of betacellulin in ovarian cancer progression are poorly understood. In the current study, we tested the hypothesis that betacellulin induces ovarian cancer cell migration by up-regulating Connexin43. Our results showed that treatment with betacellulin significantly increased Connexin43 expression and cell migration in both OVCAR4 and SKOV3 ovarian cancer cell lines. Moreover, betacellulin induced the activation of MEK-ERK signaling, and its effects on Connexin43 were inhibited by pre-treatment with U0126. Pre-treatment with AG1478 totally blocked the activation of MEK-ERK signaling but only partially inhibited betacellulin-induced Connexin43 expression and cell migration. Most importantly, betacellulin-induced cell migration was attenuated by knockdown of Connexin43, and co-treatment with gap junction inhibitor carbenoxolone did not alter this effect. Our results suggest a bilateral role of Connexin43 in ovarian cancer migration, and also demonstrate a gap junction-independent mechanism of betacellulin.

The endogenous hydrogen sulfide generating system regulates ovulation.

The generation of free-radicals such as nitric oxide has been implicated in the regulation of ovarian function, including ovulation. Tissues that generate nitric oxide typically generate another free-radical gas, hydrogen sulfide (H2S), although little is known about the role of H2S in ovarian function. The hypothesis of this study was that H2S regulates ovulation. Treatment with luteinizing hormone (LH) increased the levels of mRNA and protein of the H2S generating enzyme cystathionine γ-lyase (CTH) in granulosa cells of mice and humans in vivo and in vitro. Pharmacological inhibition of H2S generating enzymes reduced the number of follicles ovulating in mice in vivo and in vitro, and this inhibitory action was reversed by cotreatment with a H2S donor. Addition of a H2S donor to cultured mouse granulosa cells increased basal and LH-dependent abundance of mRNA encoding amphiregulin, betacellulin and tumor necrosis alpha induced protein 6, proteins important for cumulus expansion and follicle rupture. Inhibition of CTH activity reduced abundance of mRNA encoding matrix metalloproteinase-2 and -9 and tissue-type plasminogen activator, and cotreatment with the H2S donor increased the levels of these mRNA above those stimulated by LH alone. We conclude that the H2S generating system plays an important role in the propagation of the preovulatory cascade and rupture of the follicle at ovulation.

Betacellulin (BTC) Biases the EGFR To Dimerize with ErbB3.

There are 13 known endogenous ligands for the epidermal growth factor receptor (EGFR) and its closely related ErbB receptor family members. We previously reported that betacellulin (BTC) is more efficacious than epidermal growth factor (EGF) in mediating corneal wound healing, although the molecular basis for this difference was unknown. For the most part, differences between ligands can be attributed to variability in binding properties, such as the unique rate of association and dissociation, pH sensitivity, and selective binding to individual ErbB family members of each ligand. However, this was not the case for BTC. Despite being better at promoting wound healing via enhanced cell migration, BTC has reduced receptor affinity and weaker induction of EGFR phosphorylation. These data indicate that the response of BTC is not due to enhanced affinity or kinase activity. Receptor phosphorylation and proximity ligation assays indicate that BTC treatment significantly increases ErbB3 phosphorylation and EGFR-ErbB3 heterodimers when compared with EGF treatment. We observed that EGFR-ErbB3 heterodimers contribute to cell migration, because the addition of an ErbB3 antagonist (MM-121) or RNA interference-mediated knockdown of ErbB3 attenuates BTC-stimulated cell migration compared with EGF. Thus, we demonstrate that, despite both ligands binding to the EGFR, BTC biases the EGFR to dimerize with ErbB3 to regulate the biologic response.

4-Methylcatechol stimulates apoptosis and reduces insulin secretion by decreasing betacellulin and inhibin beta-A in INS-1 beta-cells.

Insulinoma INS-1 cell line is a pancreatic beta cell tumor which is characterized with high insulin content and secretion in response to increasing glucose levels. 4-Methylcatechol (4-MC) is a metabolite of quercetin, which is known as a potential drug for inhibition of tumorigenesis. The aim of this study was to determine the applying doses of 4-methylcatechol (4-MC) for triggening cell death and decreasing the cell function of rat insulinoma INS-1 beta cells. The rate of apoptosis and the amount of insulin in the cell and the secretions were determined by the ELISA method. Betacellulin (BTC) and inhibin beta-A amounts in both the cell and the glucose induced secretion were investigated by Western blotting. Furthermore, BTC, Inhibin beta-A, Ins1, Ins2, and GLUT2 gene expression levels were determined by the by the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. We noted a significant decrease in cell viability, while an increase in apoptotic cell death by 4-MC treatment. It caused a decrease in the secretion of BTC, expressions of both BTC and inhibin beta-A. We showed a decrease in the expressions of Ins1 and GLUT2, while there is no alteration in the level of insulin protein. Insulin secretion levels increased in INS-1 cells given 4-MC by basal glucose concentration while they did not response to high concentration of glucose, which indicates that 4-MC disrupts the functionality of INS-1 cells. These results revealed that 4-MC induces apoptosis and decreases insulin secretion by reducing BTC and inhibin beta-A in insulinoma INS-1 cells. Thus, 4-MC may be offered as a potential molecule for treatment of insulinoma.

Betacellulin regulates schwann cell proliferation and myelin formation in the injured mouse peripheral nerve.

When a nerve fiber is cut or crushed, the axon segment that is separated from the soma degenerates distal from the injury in a process termed Wallerian degeneration (WD). C57BL/6OlaHsd-WldS (WldS ) mutant mice exhibit significant delays in WD. This results in considerably delayed Schwann cell and macrophage responses and thus in impaired nerve regenerations. In our previous work, thousands of genes were screened by DNA microarrays and over 700 transcripts were found to be differentially expressed in the injured sciatic nerve of WldS compared with wild-type (WT) mice. One of these transcripts, betacellulin (Btc), was selected for further analysis since it has yet to be characterized in the nervous system, despite being known as a ligand of the ErbB receptor family. We show that Btc mRNA is strongly upregulated in immature and dedifferentiated Sox2+ Schwann cells located in the sciatic nerve distal stump of WT mice, but not WldS mutants. Transgenic mice ubiquitously overexpressing Btc (Tg-Btc) have increased numbers of Schmidt-Lantermann incisures compared with WT mice, as revealed by Coherent anti-Stokes Raman scattering (CARS). Tg-Btc mice also have faster nerve conduction velocity. Finally, we found that deficiency in Btc reduces the proliferation of myelinating Schwann cells after sciatic nerve injury, while Btc overexpression induces Schwann cell proliferation and improves recovery of locomotor function. Taken together, these results suggest a novel regulatory role of Btc in axon-Schwann cell interactions involved in myelin formation and nerve repair. GLIA 2017 GLIA 2017;65:657-669.

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation.

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, ß-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and ß-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and ß-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and ß-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.

Betacellulin induces Slug-mediated down-regulation of E-cadherin and cell migration in ovarian cancer cells.

Epithelial ovarian cancer is the leading cause of death among gynaecological cancers. Previous studies have demonstrated that epidermal growth factor receptor (EGFR) ligands can induce ovarian cancer cell invasion by down-regulating E-cadherin. Betacellulin is a unique member of the EGF family. It is overexpressed in a variety of cancers and is associated with reduced survival. However, the biological functions and clinical significance of betacellulin in ovarian cancer remain unknown. In the current study, we tested the hypothesis that betacellulin induces ovarian cancer cell migration by suppressing E-cadherin expression. Treatment of SKOV3 and OVCAR5 ovarian cancer cell lines with betacellulin down-regulated E-cadherin, but not N-cadherin. In addition, betacellulin treatment increased the expression of Snail and Slug, and these effects were completely blocked by pre-treatment with EGFR inhibitor AG1478. Interestingly, only knockdown of Slug reversed the down-regulation of E-cadherin by betacellulin. Betacellulin treatment induced the activation of both the MEK-ERK and PI3K-Akt signaling pathways, and it also significantly increased ovarian cancer cell migration. Importantly, the effects of betacellulin on E-cadherin, Slug and cell migration were attenuated by pre-treatment with either U0126 or LY294002. Our results suggest that betacellulin induces ovarian cancer migration and Slug-dependent E-cadherin down-regulation via EGFR-mediated MEK-ERK and PI3K-Akt signaling.

Ephrin-A5 Is Required for Optimal Fertility and a Complete Ovulatory Response to Gonadotropins in the Female Mouse.

Follicle growth and ovulation involve the coordinated expression of many genes, driven by FSH and LH. Reports indicate that Eph receptors and ephrins are expressed in the ovary, suggesting roles in follicle growth and/or ovulation. We previously reported FSH-induced expression of ephrin-A5 (EFNA5) and 4 of its cognate Eph receptors in mouse granulosa cells. We now report that female mice lacking EFNA5 are subfertile, exhibit a compromised response to LH, and display abnormal ovarian histology after superovulation. Efna5(-/-) females litters were 40% smaller than controls, although no difference in litter frequency was detected. The ovarian response to superovulation was also compromised in Efna5(-/-) females, with 37% fewer oocytes ovulated than controls. These results corresponded with a reduction in ovarian mRNA levels of several LH-responsive genes, including Pgr, Ptgs2, Tnfaip6, Ereg, Btc, and Adamts4, suggesting that Efna5(-/-) ovaries exhibit a partially attenuated response to LH. Histopathological analysis indicated that superovulated Efna5(-/-) females exhibited numerous ovarian defects, including intraovarian release of cumulus oocyte complexes, increased incidence of oocytes trapped within luteinized follicles, granulosa cell and follicular fluid emboli, fibrin thrombi, and interstitial hemorrhage. In addition, adult Efna5(-/-) ovaries exhibited a 4-fold increase in multioocyte follicles compared with controls, although no difference was detected in 3-week-old mice, suggesting the possibility of follicle merging. Our observations indicate that loss of EFNA5 in female mice results in subfertility and imply that Eph-ephrin signaling may also play a previously unidentified role in the regulation of fertility in women.

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin.

Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically (156)EEMETL(161)). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization.

Growth Factor Identity Is Encoded by Discrete Coiled-Coil Rotamers in the EGFR Juxtamembrane Region.

Binding of transforming growth factor α (TGF-α) to the epidermal growth factor receptor (EGFR) extracellular domain is encoded through the formation of a unique antiparallel coiled coil within the juxtamembrane segment. This new coiled coil is an "inside-out" version of the coiled coil formed in the presence of epidermal growth factor (EGF). A third, intermediary coiled-coil interface is formed in the juxtamembrane region when EGFR is stimulated with betacellulin. The seven growth factors that activate EGFR in mammalian systems (EGF, TGF-α, epigen, epiregulin, betacellulin, heparin-binding EGF, and amphiregulin) fall into distinct categories in which the structure of the coiled coil induced within the juxtamembrane region correlates with cell state. The observation that coiled-coil state tracks with the downstream signaling profiles for each ligand provides evidence for growth factor functional selectivity by EGFR. Encoding growth factor identity in alternative coiled-coil rotamers provides a simple and elegant method for communicating chemical information across the plasma membrane.

Betacellulin transgenic mice develop urothelial hyperplasia and show sex-dependent reduction in urinary major urinary protein content.

The epidermal growth factor (EGF)-like ligands and their cognate ERBB1-4 receptors represent important signaling pathways that regulate tissue and cell proliferation, differentiation and regeneration in a wide variety of tissues, including the urogenital tract. Betacellulin (BTC) can activate all four ERBB tyrosine kinase receptors and is a multifunctional EGF-like ligand with diverse roles in ß cell differentiation, bone maturation, formation of functional epithelial linings and vascular permeability in different organs. Using transgenic BTC mice, we have studied the effect of constitutive systemic BTC over-expression on the urinary bladder. BTC was detected in microvascular structures of the stromal bladder compartment and in umbrella cells representing the protective apical lining of the uroepithelium. ERBB1 and ERBB4 receptors were co-localized in the urothelium. Mice transgenic for BTC and double transgenic for both BTC and the dominant kinase-dead mutant of EGFR (Waved 5) developed hyperplasia of the uroepithelium at 5months of age, suggesting that urothelial hyperplasia was not exclusively dependent on ERBB1/EGFR. Mass spectrometric analysis of urine revealed a significant down-regulation of major urinary proteins in female BTC transgenic mice, suggesting a novel role for systemic BTC in odor-based signaling in female transgenic BTC mice.