Dysregulation of the NRG1/ERBB pathway causes a developmental disorder with gastrointestinal dysmotility in humans.

Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system, with an incidence of 1 in 5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle-specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in 8 individuals variably associating HSCR, CIPO, peripheral neuropathy, and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis, and intestinal smooth muscle abnormalities. The cell type-specific ErbB3 and ErbB2 function was further analyzed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using quantitative real-time PCR (RT-qPCR) on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing WT or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies, including intestinal dysmotility.

CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes.

While the epidermal growth factor receptor (EGFR) is an established regulator of skin development and homeostasis, the functions of the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue have only recently been examined. Previously reported, skin-specific deletion of each of these receptors in mice resulted in similar defects in keratinocyte proliferation and migration, resulting in impaired wound healing and tumorigenesis. Because both ERBB2 and ERBB3 are targets for treating an array of cancer types, it is important to examine the consequences of receptor inhibition in human keratinocytes. Here, we employed the CRISPR/Cas9 technology to generate HaCaT cells (an established human keratinocyte cell line) lacking ERBB2 or ERBB3. HaCaT clones lacking ERBB2 or ERBB3 showed comparable reductions in cell proliferation as assessed by BrdU staining. Apoptosis, in contrast, was reduced in ERBB3-deficient HaCaT cells only. Assessment of cell migration using a wound healing (scratch) assay showed that the closure of the wound gaps was completed by 48 h in mock and in ERBB3 knockout clones. In contrast, this process was considerably delayed in ERBB2 knockout clones, and a complete closure of the gap in the latter cells did not occur before 72 h. In conclusion, both ERBB2 and ERBB3 are essential for normal proliferation of skin keratinocytes, but in contrast to ERBB3, ERBB2 is essential for migration of human keratinocytes. These observations might bear significance to patient adverse effects of therapeutic agents targeting ERBB2 and ERBB3.

The ErbB3 receptor tyrosine kinase negatively regulates Paneth cells by PI3K-dependent suppression of Atoh1.

Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1+ cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.

Loss of hepatocyte ERBB3 but not EGFR impairs hepatocarcinogenesis.

Epidermal growth factor receptor (EGFR) and ERBB3 have been implicated in hepatocellular carcinogenesis (HCC). However, it is not known whether altering the activity of either EGFR or ERBB3 affects HCC development. We now show that Egfr(Dsk5) mutant mice, which have a gain-of-function allele that increases basal EGFR kinase activity, develop spontaneous HCC by 10 mo of age. Their tumors show increased activation of EGFR, ERBB2, and ERBB3 as well as AKT and ERK1,2. Hepatocyte-specific models of EGFR and ERBB3 gene ablation were generated to evaluate how the loss of these genes affected tumor progression. Loss of either receptor tyrosine kinase did not alter liver development or regenerative liver growth following carbon tetrachloride injection. However, using a well-characterized model of HCC in which N-nitrosodiethylamine is injected into 14-day-old mice, we discovered that loss of hepatocellular ERBB3 but not EGFR, which occurred after tumor initiation, retarded liver tumor formation and cell proliferation. We found no evidence that this was due to increased apoptosis or diminished phosphatidylinositol-3-kinase activity in the ERBB3-null cells. However, the relative amount of phospho-STAT3 was diminished in tumors derived from these mice, suggesting that ERBB3 may promote HCC through STAT3 activation.

Neuregulin-1ß induces embryonic stem cell cardiomyogenesis via ErbB3/ErbB2 receptors.

NRG-1ß (neuregulin-1ß) serves multiple functions during embryonic heart development by signalling through ErbB family receptor tyrosine kinases (ErbB2, ErbB3 and ErbB4). Previous studies reported that NRG-1ß induces cardiomyogenesis of mESCs (mouse embryonic stem cells) at the later stages of differen-tiation through ErbB4 receptor activation. In the present study we systematically examined NRG-1ß induction of cardiac myocytes in mESCs and identified a novel time window, the first 48 h, for NRG-1ß-based cardiomyogenesis. At this time point ErbB3, but not ErbB4, is expressed. In contrast with the later differentiation of mESCs in which NRG-1ß induces cardiomyogenesis via the ErbB4 receptor, we found that knocking down ErbB3 or ErbB2 with siRNA during the early differentiation inhibited NRG-1ß-induced cardiomyogenesis in mESCs. Microarray analysis of RNA expression at this early time point indicated that NRG-1ß treatment in mESCs resulted in gene expression changes important to differentiation including up-regulation of components of PI3K (phosphoinositide 3-kinase), a known mediator of the NRG-1ß/ErbB signalling pathway, as well as activation of CREB (cAMP-response-element-binding protein). Further study demonstrated that the NRG-1ß-induced phosphorylation of CREB was required for cardiomyogenesis of mESCs. In summary, we report a previously unrecognized role for NRG-1ß/ErbB3/CREB signalling at the pre-mesoderm stage for stem cell cardiac differentiation.

Selective regulation of nerve growth factor expression in developing cutaneous tissue by early sensory innervation.

BACKGROUND: In the developing vertebrate peripheral nervous system, the survival of sympathetic neurons and the majority of sensory neurons depends on a supply of nerve growth factor (NGF) from tissues they innervate. Although neurotrophic theory presupposes, and the available evidence suggests, that the level of NGF expression is completely independent of innervation, the possibility that innervation may regulate the timing or level of NGF expression has not been rigorously investigated in a sufficiently well-characterized developing system. RESULTS: To address this important question, we studied the influence of innervation on the regulation of NGF mRNA expression in the embryonic mouse maxillary process in vitro and in vivo. The maxillary process receives its innervation from predominantly NGF-dependent sensory neurons of the trigeminal ganglion and is the most densely innervated cutaneous territory with the highest levels of NGF in the embryo. When early, uninnervated maxillary processes were cultured alone, the level of NGF mRNA rose more slowly than in maxillary processes cultured with attached trigeminal ganglia. In contrast to the positive influence of early innervation on NGF mRNA expression, the levels of brain-derived neurotrophic factor (BDNF) mRNA and neurotrophin-3 (NT3) mRNA rose to the same extent in early maxillary processes grown with and without trigeminal ganglia. The level of NGF mRNA, but not BDNF mRNA or NT3 mRNA, was also significantly lower in the maxillary processes of erbB3-/- mice, which have substantially fewer trigeminal neurons than wild-type mice. CONCLUSIONS: This selective effect of initial innervation on target field NGF mRNA expression provokes a re-evaluation of a key assertion of neurotrophic theory that the level of NGF expression is independent of innervation.

ErbB3 ablation impairs PI3K/Akt-dependent mammary tumorigenesis.

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling.

Tumor-specific apoptosis caused by deletion of the ERBB3 pseudo-kinase in mouse intestinal epithelium.

Pharmacologic blockade of EGFR or the closely related receptor ERBB2 has modest efficacy against colorectal cancers in the clinic. Although the upregulation of ERBB3, a pseudo-kinase member of the EGFR/ERBB family, is known to contribute to EGFR inhibitor resistance in other cancers, its functions in normal and malignant intestinal epithelium have not been defined. We have shown here that the intestinal epithelium of mice with intestine-specific genetic ablation of Erbb3 exhibits no cytological abnormalities but does exhibit loss of expression of ERBB4 and sensitivity to intestinal damage. By contrast, intestine-specific Erbb3 ablation resulted in almost complete absence of intestinal tumors in the ApcMin mouse model of colon cancer. Unlike nontransformed epithelium lacking ERBB3, intestinal tumors lacking ERBB3 had reduced PI3K/AKT signaling, which led to attenuation of tumorigenesis via a tumor-specific increase in caspase-3-mediated apoptosis. Consistent with the mouse data, which suggest that ERBB3-ERBB4 heterodimers contribute to colon cancer survival, experimentally induced loss of ERBB3 in a KRAS mutant human colon cancer cell line was associated with loss of ERBB4 expression, and siRNA knockdown of either ERBB3 or ERBB4 resulted in elevated levels of apoptosis. These results indicate that the ERBB3 pseudo-kinase has essential roles in supporting intestinal tumorigenesis and suggest that ERBB3 may be a promising target for the treatment of colorectal cancers.

erbB3 is dispensable for oligodendrocyte development in vitro and in vivo.

During development and in the adult, erbB2, erbB3, and erbB4 are expressed in many tissues and as heterodimers (B2/B3, B2/B4) serve as receptors for neuregulins. The general importance of neuregulin receptors for development is underlined by the observed embryonic (erbB2, erbB4) or perinatal (erbB3) lethality in mouse mutants. These mutants further revealed the fundamental role of the erbB2/erbB3 heterodimer for proper Schwann cell development, the ensheathing glia of the peripheral nervous system. However, only little is known about the functions of neuregulins and their receptors during postnatal development and in the adult. erbB2 and erbB3 during late embryogenesis and postnatally are expressed in different areas and cell types of the central nervous system, including oligodendrocytes, the ensheathing glia of the central nervous system. As terminal differentiation of oligodendrocytes peaks during postnatal development, it is not possible to use the neuregulin receptor mouse mutants to study terminal differentiation of oligodendrocytes in their absence in vivo. In order to investigate possible functions of the erbB3 gene in oligodendrocytes, we employed two different techniques. First, we directed the differentiation of erbB3-deficient embryonic stem cells into neural cell types to analyze the development of oligodendrocytes in the absence of erbB3 in vitro. Second, we grafted neural stem cells from spinal cords of erbB3 mutants into the retina of young mice to monitor oligodendrocyte differentiation and myelination in vivo. Results of both experimental approaches clearly show that erbB3 is not required for normal oligodendrocyte development and myelination.

Heregulin, but not ErbB2 or ErbB3, heterozygous mutant mice exhibit hyperactivity in multiple behavioral tasks.

Genetic redundancy is a problem in gene targeting studies because functionally relevant sister proteins can compensate for the lack of protein product of a targeted gene. A molecular system is chosen in which it is hoped to demonstrate both the lack and presence of compensation after disruption of particular single genes. Mammals may not be able to compensate for the lack of heregulin, a single ligand for multiple ErbB receptors, however, compensation is expected when a single ErbB receptor is knocked out. To investigate this the heregulin-1, ErbB2, or ErbB3 locus was disrupted in a targeted manner and mice heterozygous for the mutation were analyzed. Heregulin and its receptors were shown to be involved in embryonic brain development and, more recently, in plastic changes associated with adult brain function in rodents. Although they have never been shown to play roles in mammalian behavior, it was decided to characterize the mice behaviorally using a battery of simple tests. Heregulin mutant mice exhibited elevated activity levels in the open field, showed improved rotorod performance, and finished T-maze spontaneous alternation task faster compared to control wild type littermates, findings that suggest a consistent hyperactivity across tests. ErbB2 and ErbB3 mutant mice, whose strain origin was identical to that of heregulin mutants, showed no sign of the behavioral alterations. It is suggested that the abnormalities seen in heregulin mutant mice are due to mutation at that locus and the lack of alterations seen in ErbB2 and ErbB3 mutant mice is the result of compensation by unaltered sister receptors.