Source and Impact of the EGF Family of Ligands on Intestinal Stem Cells

Epidermal Growth Factor (EGF) has long been known for its role in promoting proliferation of intestinal epithelial cells. EGF is produced by epithelial niche cells at the base of crypts in vivo and is routinely added to the culture medium to support the growth of intestinal organoids ex vivo. The recent identification of diverse stromal cell populations that reside underneath intestinal crypts has enabled the characterization of key growth factor cues supplied by these cells. The nature of these signals and how they are delivered to drive intestinal epithelial development, daily homeostasis and tissue regeneration following injury are being investigated. It is clear that aside from EGF, other ligands of the family, including Neuregulin 1 (NRG1), have distinct roles in supporting the function of intestinal stem cells through the ErbB pathway.

Dopamine System, NMDA Receptor and EGF Family Expressions in Brain Structures of Bl6 and 129Sv Strains Displaying Different Behavioral Adaptation

C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv) mice display different coping strategies in stressful conditions. Our aim was to evaluate biomarkers related to different adaptation strategies in the brain of male 129Sv and Bl6 mice. We focused on signaling pathways related to the dopamine (DA) system, N-methyl-D-aspartate (NMDA) receptor and epidermal growth factor (EGF) family, shown as the key players in behavioral adaptation. Mice from Bl6 and 129Sv lines were divided into either home cage controls (HCC group) or exposed to repeated motility testing and treated with saline for 11 days (RMT group). Distinct stress responses were reflected in severe body weight loss in 129Sv and the increased exploratory behavior in Bl6 mice. Besides that, amphetamine caused significantly stronger motor stimulation in Bl6. Together with the results from gene expression (particularly Maob), this study supports higher baseline activity of DA system in Bl6. Interestingly, the adaptation is reflected with opposite changes of DA markers in dorsal and ventral striatum. In forebrain, stress increased the gene expressions of Egf-Erbb1 and Nrg1/Nrg2-Erbb4 pathways more clearly in 129Sv, whereas the corresponding proteins were significantly elevated in Bl6. We suggest that not only inhibited activity of the DA system, but also reduced activity of EGF family and NMDA receptor signaling underlies higher susceptibility to stress in 129Sv. Altogether, this study underlines the better suitability of 129Sv for modelling neuropsychiatric disorders than Bl6.

ErbB Receptor Stimulation Is Required for Mouse Colon Adenoma Organoids to Form Crypts

The majority of colon adenomas harbor genetic mutations in the APC gene. APC mutation leads to changes in Wnt signalling and cell-cell adhesion: as a consequence, intestinal crypt budding increases and the excess crypts accumulate to form adenomas that progress to colon cancer. When cultured with Wnt, R-spondin, EGF, Noggin, myofibroblast conditioned medium and Matrigel, crypts from normal mouse colon mucosa form crypt-producing organoids and can be passaged continuously. Under the same culture and passage conditions, crypts isolated from colon adenomas derived from Apcmin/+ mice typically grow as spheroidal cysts and do not produce crypts. The adenoma organoid growth requires EGF, but not Wnt, R-spondin or Noggin. However, when mouse colon adenoma spheroids are grown for more than 10 days in the presence of EGF, crypt formation occurs. EGF, EREG, β-cellulin, Neuregulin-1 or AREG are sufficient for initiating crypt formation, however, neuregulin-1 is more potent than the other EGF-family members. EGFR and ErbB2 inhibitors both prevent crypt formation in adenoma cultures. Either EGFR:ErbB2 or ErbB3:ErbB2 signalling is sufficient to initiate adenoma crypt budding and elongation. ErbB2 inhibitors may provide a therapeutic avenue for controlling and ablating colon adenomas.

Compatibility of intravitreally applied epidermal growth factor and amphiregulin

Introduction To examine the compatibility of intravitreally injected epidermal growth factor (EGF) and amphiregulin as EGF family member. Methods Four rabbits (age: 4 months; body weight: 2.5 kg) received three intravitreal injections of EGF (100 ng) uniocularly in monthly intervals and underwent ocular photography, tonometry, biometry, and optical coherence tomography. After sacrificing the rabbits, the globes were histomorphometrically examined. In a second study part, eyes of 22 guinea pigs (age: 2–3 weeks) received two intravitreal administrations of amphiregulin (10 ng) or phosphate buffered solution (PBS) in 10-day interval, or were left untouched. Ten days after the second injection, the guinea pigs were sacrificed, the enucleated eyes underwent histological and immune-histological examinations. Results The rabbit eyes with EGF injections versus the contralateral untouched eyes did not show significant differences in intraocular pressure (7.5 ± 2.4 mmHg vs. 6.8 ± 2.2 mmHg; P = 0.66), retinal thickness (158 ± 5 µm vs. 158 ± 3 µm; P = 1.0), cell counts in the retinal ganglion cell layer (3.3 ± 1.7 cells/150 µm vs. 3.0 ± 1.4 cells/150 µm; P = 0.83), inner nuclear layer (46.4 ± 23.2 cells/150 µm vs. 39.6 ± 6.4 cells/150 µm; P = 0.61), and outer nuclear layer (215 ± 108 cells/150 µm vs. 202 ± 47 cells/150 µm; P = 0.83), or any apoptotic retinal cells. The guinea pig eyes injected with amphiregulin versus eyes with PBS injections did not differ (P = 0.72) in the degree of microglial activation, and both groups did not differ from untouched eyes in number of apoptotic retinal cells and retinal gliosis. Conclusions Intravitreal applications of EGF (100 ng) in rabbits nor intravitreal applications of amphiregulin (10 ng) in guinea pigs led to intraocular specific inflammation or any observed intraocular destructive effect. The findings support the notion of a compatibility of intraocular applied EGF and amphiregulin.

Epiregulin (EREG) and Myocardin Related Transcription Factor A (MRTF-A) Form a Feedforward Loop to Drive Hepatic Stellate Cell Activation

Trans-differentiation of quiescent hepatic stellate cells (HSC) into myofibroblast cells is considered the linchpin of liver fibrosis. A myriad of signaling pathways contribute to HSC activation and consequently liver fibrosis. Epidermal growth factor (EGF) family of cytokines signal through the cognate receptor EGFR to promote HSC activation. In the present study we investigated the transcription regulation of epiregulin (EREG), an EGFR ligand, during HSC activation. We report that EREG expression was significantly up-regulated in activated HSCs compared to quiescent HSCs isolated from mice. In addition, there was an elevation of EREG expression in HSCs undergoing activation in vitro. Of interest, deficiency of myocardin-related transcription factor A (MRTF-A), a well-documented regulator of HSC trans-differentiation, attenuated up-regulation of EREG expression both in vivo and in vitro. Further analysis revealed that MRTF-A interacted with serum response factor (SRF) to bind directly to the EREG promoter and activate EREG transcription. EREG treatment promoted HSC activation in vitro, which was blocked by MRTF-A depletion or inhibition. Mechanistically, EREG stimulated nuclear trans-location of MRTF-A in HSCs. Together, our data portray an EREG-MRTF-A feedforward loop that contributes to HSC activation and suggest that targeting the EREG-MRTF-A axis may yield therapeutic solutions against liver fibrosis.

Sustained ErbB activation causes demyelination and hypomyelination by driving necroptosis of mature oligodendrocytes and apoptosis of oligodendrocyte precursor cells

Oligodendrocytes are vulnerable to genetic and environmental insults and its injury leads to demyelinating diseases. The roles of ErbB receptors in the CNS myelin integrity are largely unknown. Here we overactivate ErbB receptors that mediate signaling of either neuregulin or EGF family growth factors and found their synergistic activation caused deleterious outcomes in white matter. Sustained ErbB activation induced by the tetracycline-dependent mouse tool Plp-tTA resulted in demyelination, axonal degeneration, oligodendrocyte precursor cell (OPC) proliferation, astrogliosis, and microgliosis in white matter. Moreover, there was hypermyelination prior to these pathological events. In contrast, sustained ErbB activation induced by another tetracycline-dependent mouse tool Sox10+/rtTA caused hypomyelination in the corpus callosum and optic nerve, which appeared to be a developmental deficit and did not associate with OPC regeneration, astrogliosis, or microgliosis. By analyzing the differentiation states of cells that were pulse- labeled with a viral reporter, we found that, during juvenile to adolescent development, Plp-tTA targeted mainly mature oligodendrocytes (MOs), while Sox10+/rtTA targeted OPCs and newly-formed oligodendrocytes. The distinct phenotypes of mice with ErbB overactivation induced by Plp-tTA and Sox10+/rtTA supported the reporter pulse-labeling results, and consolidated their non-overlapping targeting preferences in the oligodendrocyte lineage after early development. These features enabled us to demonstrate that ErbB overactivation in MOs induced necroptosis that caused pathological demyelination, whereas in OPCs induced apoptosis that caused developmental hypomyelination. These results established an upstream pathogenic role of ErbB overactivation in oligodendrocytes, providing molecular and cellular insights into the primary oligodendropathy in demyelinating diseases.

Baboon placental heparin-binding epidermal growth factor-like growth factor

Placental extravillous trophoblast remodeling of the uterine spiral arteries is important for promoting blood flow to the placenta and fetal development. Heparin-binding EGF-like growth factor (HB-EGF), an EGF family member, stimulates differentiation and invasive capacity of extravillous trophoblasts in vitro. Trophoblast expression and maternal levels of HB-EGF are reduced at term in women with preeclampsia, but it is uncertain whether HB-EGF is downregulated earlier when it may contribute to placental insufficiency. A nonhuman primate model has been established in which trophoblast remodeling of the uterine spiral arteries is suppressed by shifting the rise in estrogen from the second to the first trimester of baboon pregnancy. In the present study, we used this model to determine if placental HB-EGF is altered by prematurely elevating estrogen early in baboon gestation. Uterine spiral artery remodeling and placental expression of HB-EGF and other EGF family members were assessed on day 60 of gestation in baboons treated with estradiol (E2) daily between days 25 and 59 of gestation (term = 184 days). The percentages of spiral artery remodeling were 90, 84 and 70% lower (P < 0.01), respectively, for vessels of 26–50, 51–100 and >100 µm diameter in E2-treated compared with untreated baboons. HB-EGF protein quantified by immunocytochemical staining/image analysis was decreased three-fold (P < 0.01) in the placenta of E2-treated versus untreated baboons, while amphiregulin (AREG) and EGF expression was unaltered. Therefore, we propose that HB-EGF modulates the estrogen-sensitive remodeling of the uterine spiral arteries by the extravillous trophoblast in early baboon pregnancy.

Proteolytic Processing of Neuregulin 2

Neuregulin 2 (NRG2) belongs to the EGF family of growth factors. Most of this family members require proteolytic cleavage to liberate their ectodomains capable of binding and activating their cognate ErbB receptors. To date, most of the studies investigating proteolytic processing of neuregulins focused on NRG1, which was shown to undergo ectodomain shedding by several ADAM proteases and BACE1 and the remaining fragment was further cleaved by γ-secretase. Recently, NRG2 attracted more attention due to its role in the neurogenesis and modulation of behaviors associated with psychiatric disorders. In this study, we used genetic engineering methods to identify proteases involved in proteolytic processing of murine NRG2. Using non-neuronal cell lines as well as cultures of primary hippocampal neurons, we demonstrated that the major proteases responsible for releasing NRG2 ectodomain are ADAM10 and BACE2. Co-expression of NRG2 and BACE2 in neurons of certain brain structures including medulla oblongata and cerebellar deep nuclei was confirmed via immunohistochemical staining. The cleavage of NRG2 by ADAM10 or BACE2 generates a C-terminal fragment that serves as a substrate for γ-secretase. We also showed that murine NRG2 is subject to post-translational modifications, substantial glycosylation of its extracellular part, and phosphorylation of the cytoplasmic tail.

Anti-Factor XII Autoantibodies in Patients with Recurrent Pregnancy Loss Recognize the Second Epidermal Growth Factor–Like Domain

Background Factor XII (FXII) deficiency and autoantibodies that bind to FXII (anti-FXII) have been described in patients with adverse pregnancy outcomes, including recurrent pregnancy loss. It has been reported that FXII functions not only as a coagulation protein but also as a growth factor. Objectives We studied the association between anti-FXII and the epidermal growth factor (EGF) system in patients with recurrent pregnancy loss. Patients/Methods We used synthetic peptides that span the second EGF-like domain in the heavy chain of FXII (EGF2) in inhibition and direct binding studies to determine if anti-FXII antibodies recognize EGF2. Furthermore, we examined whether anti-FXII antibodies, which recognize EGF2, also recognize recombinant EGF and heparin-binding EGF-like growth factor (HB-EGF). Results Among 100 patients with recurrent pregnancy loss, the plasma of 23 patients (23.0%) recognized the synthetic peptide ASQ41, which covers EGF2. Among the 23 anti-ASQ41-positive patients, plasma samples from 13 patients (56.5%) recognized the 22-residue segment C-terminal half of ASQ41. Among the 23 anti-ASQ41-positive patients, the plasma of 17 patients (73.9%) recognized recombinant human EGF. Affinity-purified anti-FXII antibodies, which recognize ASQ41, also recognized recombinant EGF family proteins such as EGF and HB-EGF. Conclusions The autoantibodies in patients with recurrent pregnancy loss recognized the EGF2 domain in FXII and other proteins of the EGF family. Since proteins in the EGF family play an important role in normal pregnancy, autoantibody-associated disruption of the EGF system may cause pregnancy loss.

Elevated serum neuregulin 4 levels in patients with hyperthyroidism

Objective Recent studies have shown that neuregulin 4 (Nrg4), a member of the epidermal growth factor (EGF) family of extracellular ligands, plays an important role in the prevention of obesity, insulin resistance and nonalcoholic fatty liver disease (NAFLD). Considering that thyroid hormone (TH) has profound effects on whole-body energy metabolism, we speculate that circulating Nrg4 levels might be altered in patients with hyperthyroidism. Design and methods A total of 129 hyperthyroid patients and 100 healthy subjects were recruited. Of them, 39 hyperthyroid patients received thionamide treatment for 3 months until euthyroidism. Serum Nrg4 levels were determined using the ELISA method. To further confirm the relationship between TH and Nrg4, C57BL/6 mice were treated with T3 and quantitative real-time PCR was performed to detect Nrg4 gene expression. Results Serum Nrg4 levels were significantly elevated in hyperthyroid patients as compared with normal controls (3.84 ± 1.63 vs 2.21 ± 1.04 ng/mL, P < 0.001). After achieving euthyroidism by thionamide treatment, serum Nrg4 levels dropped markedly from 3.57 ± 1.26 to 1.94 ± 0.72 ng/ml (P < 0.001). After adjustment for potential confounders, serum Nrg4 levels were independently associated with hyperthyroidism. The upregulation of Nrg4 expression in the livers and white adipose tissues by T3 was further confirmed by animal and cell culture experiments. Conclusions Serum Nrg4 levels were increased in patients with hyperthyroidism. The liver and white adipose tissue might be primary sources contributing to elevated serum Nrg4 concentrations.