MUC4 involvement in ErbB2/ErbB3 phosphorylation and signaling in response to airway cell mechanical injury

The receptor tyrosine kinases ErbB2 and ErbB3 are phosphorylated in response to injury of the airway epithelium. Since we have shown that the membrane mucin MUC4 can act as a ligand/modulator for ErbB2, affecting its localization in polarized epithelial cells and its phosphorylation, we questioned whether Muc4 was involved, along with ErbB2 and ErbB3, in the damage response of airway epithelia. To test this hypothesis, we first examined the localization of MUC4 in human airway samples. Both immunocytochemistry and immunofluorescence showed a co‐localization of MUC4 and ErbB2 at the airway luminal surface. Sequential immunoprecipitation and immunoblotting from airway cells demonstrated that the MUC4 and ErbB2 are present as a complex in airway epithelial cells. To assess the participation of MUC4 in the damage response, cultures of NCI‐H292 or airway cells were scratch‐wounded, then analyzed for association of phospho‐ErbB2 and ‐ErbB3 with MUC4 by sequential immunoprecipitation and immunoblotting. Wounded cultures exhibited increased phosphorylation of both receptors in complex with MUC4. Scratch wounding also increased activation of the downstream pathway through Akt, as predicted from our previous studies on Muc4 effects on ErbB2 and ErbB3. The participation of MUC4 in the phosphorylation response was also indicated by siRNA repression of MUC4 expression, which resulted in diminution of the phosphorylation of ErbB2 and ErbB3. These studies provide a new model for the airway epithelial damage response, in which the MUC4–ErbB2 complex is a key element in the sensor mechanism and phosphorylation of the receptors. J. Cell. Biochem. 107: 112–122, 2009. © 2009 Wiley‐Liss, Inc.     

ErbB signaling in cardiac development and disease

The ErbB family of receptor tyrosine kinases (RTKs) is a family of receptors that allow cells to interact with the extracellular environment and transduce signals to the nucleus that promote differentiation, migration and proliferation necessary for proper heart morphogenesis and function. This review focuses on the role of the ErbB family of receptor tyrosine kinases, and their importance in proper heart morphogenesis, as well as their role in maintenance and function of the adult heart. Studies from transgenic mouse models have shown the importance of ErbB receptors in heart development, and provide insight into potential future therapeutic targets to help reduce congenital heart defect (CHD) mortality rates and prevent disease in adults. Cancer therapeutics have also shed light to the ErbB receptors and signaling network, as undesired side effects have demonstrated their importance in adult cardiomyocytes and prevention of cardiomyopathies. This review will discuss ErbB receptor tyrosine kinases (RTK) in heart development and disease including valve formation and partitioning of a four-chambered heart as well as cardiotoxicity when ErbB signaling is attenuated in adults.     

The role of Neuregulin-1beta/ErbB signaling in the heart

Products of the Neuregulin-1 (Nrg-1) gene, along with the ErbB family of receptor tyrosine kinases through which Nrg-1 ligands signal, play a critical role during cardiovascular development. Through studies of genetically manipulated mice, as well as studies in cells isolated from adult hearts, it appears that Nrg-1/ErbB signaling is an essential paracrine mediator of cell-cell interactions that not only regulates tissue organization during development, but also helps to maintain cardiac function throughout an organism's life. Studies in cells isolated from the heart demonstrate that Nrg-1 can activate a number of signaling pathways, which mediate cellular adaptations to stress in the myocardium. These observations provide insight as to why ErbB2-targeted cancer treatments have deleterious effects on cardiac function in some cancer patients. Moreover emerging data suggest that Nrg-1 ligands might be useful clinically to restore cardiac function after cardiac injury. In this review we will attempt to synthesize the literature behind this rapidly growing and exciting area of research.     

Identification of prolidase as a high affinity ligand of the ErbB2 receptor and its regulation of ErbB2 signaling and cell growth

ErbB2, an important membrane-bound receptor tyrosine kinase, was discovered nearly 30 years ago, but a natural ligand has never been found previously. ErbB2 is also an important oncogene and anticancer target, and its overexpression in cancer is associated with poor disease prognosis. Here, we report that human prolidase (PEPD) is a high affinity ligand of ErbB2 and binds as a homodimer to subdomain 3 in the extracellular domain of this receptor. In ErbB2-overexpressing cells, both ErbB2 monomers and activated dimers exist. PEPD bound to ErbB2 monomers relatively slowly but caused ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. In contrast, PEPD bound rapidly to ErbB2 homodimers and rapidly silenced ErbB2 dimer-Src signaling, a key oncogenic pathway of ErbB2, by disrupting the association of Src with ErbB2. PEPD also caused pronounced ErbB2 depletion, resulting from ErbB2 internalization and degradation. Moreover, PEPD strongly inhibited the DNA synthesis, anchorage-independent growth and invasion and migration of cells that overexpressed ErbB2 but had no effect on cells without ErbB2 overexpression. Cells became sensitized to PEPD upon achieving stable ErbB2 overexpression. Thus, the impact of PEPD on ErbB2 is predominantly inhibitory, and PEPD targets cells addicted to ErbB2. PEPD is also a dipeptidase, but its enzymatic function is not involved in ErbB2 modulation. These findings revise our understanding of ErbB2 and PEPD and may be especially important for combating ErbB2-positive cancers.     

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier

Background

Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs).

Methods

Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1.

Results

HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRG's effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA.

Conclusion

These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.     

Effects of inflammatory cytokines on bone/cartilage repair

Many inflammatory factors can affect cell behaviors and work as a form of inter-regulatory networks through the inflammatory pathway. Inflammatory cytokines are critical for triggering bone regeneration after fracture or bone injury. Also, inflammatory cytokines play an important role in cartilage repair. The synergistic or antagonistic effects of both proinflammatory and anti-inflammatory cytokines have a great influence on fracture healing. This review discusses key inflammatory cytokines and signaling pathways involved in bone or cartilage repair.     

TGFβ signaling in cartilage development and maintenance

Members of the transforming growth factor beta (TGFβ) superfamily of secreted factors play essential roles in nearly every aspect of cartilage formation and maintenance. However, the mechanisms by which TGFβs transduce their effects in cartilage in vivo remain poorly understood. Mutations in several TGFβ family members, their receptors, extracellular modulators, and intracellular transducers have been described, and these usually impact the development of the cartilaginous skeleton. Furthermore, genome‐wide association studies have linked components of the (TGFβ) superfamily to susceptibility to osteoarthritis. This review focuses on recent discoveries from genetic studies in the mouse regarding the regulation of TGFβ signaling in developing growth plate and articular cartilage, as well as the different modes of crosstalk between canonical and noncanonical TGFβ signaling. These new insights into TGFβ signaling in cartilage may open new prospects for therapies that maintain healthy articular cartilage. Birth Defects Research (Part C) 102:37–51, 2014. © 2014 Wiley Periodicals, Inc.     

Crosstalk between the extracellular domain of the ErbB2 receptor and IGF-1 receptor signaling

Insulin-like growth factor 1 receptor (IGF-1R) plays an important role in cell growth and malignant transformation. To investigate IGF-1R-dependent signaling events and its effects on apoptosis induction and cellular proliferation, we generated a constitutively active, ligand-independent IGF-1R variant. We fused the cytoplasmic domain of the IGF-1R to the extracellular and transmembrane domains of the oncogenic ErbB2 receptor (ErbB2^V→E/IGF-1). A fusion protein in which the wild-type sequence of the ErbB2 receptor was used, served as a control (ErbB2^V/IGF-1R). ErbB2^V/IGF-1R, ErbB2^V→E/IGF-1R and IGF-1R were stably transfected into interleukin 3 (IL-3)-dependent BaF/3 cells. ErbB2^V→E/IGF-1R expressing cells exhibited ligand-independent, constitutive tyrosine phosphorylation of the receptor fusion protein. Constitutively, activated ErbB2^V→E/IGF-1R conferred IL-3 independence for growth and survival to the transfected BaF/3 cells. Constitutive activation of the IGF-1R results in cellular growth and protection against apoptosis upon IL-3 withdrawal in BaF/3 cells.     

Inhibition of ErbB2/neuregulin signaling augments paclitaxel-induced cardiotoxicity in adult ventricular myocytes

Paclitaxel (Taxol) has been successfully combined with the monoclonal antibody trastuzumab (Herceptin) in the treatment of ErbB2 overexpressing cancers. However, this combination therapy showed an unexpected synergistic increase in cardiac dysfunction. We have studied the mechanisms of paclitaxel/anti-ErbB2 cardiotoxicity in adult rat ventricular myocytes (ARVM). Myofibrillar organization was assessed by immunofluorescence microscopy and cell viability was tested by the TUNEL-, LDH- and MTT-assay. Oxidative stress was measured by DCF-fluorescence and myocyte contractile function by video edge-detection and fura-2 fluorescence. Treatment of ARVM with paclitaxel or antibodies to ErbB2 caused a significant increase in myofilament degradation, similarly as observed with an inhibitor of MAPK-signaling, but not apoptosis, necrosis or changes in mitochondrial activity. Paclitaxel-treatment and anti-ErbB2 reduced Erk1/2 phosphorylation. Paclitaxel increased diastolic calcium, shortened relaxation time and reduced fractional shortening in combination with anti-ErbB2. A minor increase in oxidative stress by paclitaxel or anti-ErbB2 was found. We conclude, that concomitant inhibition of ErbB2 receptors and paclitaxel treatment has an additive worsening effect on adult cardiomyocytes, mainly discernible in changes of myofibrillar structure and function, but in the absence of cell death. A potential mechanism is the modulation of the MAPK/Erk1/2 signaling by both drugs.     

Integrin signaling in skeletal development and function

Integrins are cell surface receptors that connect extracellular matrix (ECM) components to the actin cytoskeleton and transmit chemical and mechanical signals into the cells through adhesion complexes. Integrin‐activated downstream pathways have been implicated in the regulation of various cellular functions, including proliferation, survival, migration, and differentiation. Integrin‐based attachment to the matrix plays a central role in development, tissue morphogenesis, adult tissue homeostasis, remodeling and repair, and disturbance of the ECM‐integrin‐cytoskeleton signaling axis often results in diseases and tissue dysfunction. Increasing amount of in vitro and in vivo evidences suggest that integrins are pivotal for proper development, function, and regeneration of skeletal tissues. In this paper, we will summarize and discuss the role of integrins in skeletogenesis and their influence on the physiology and pathophysiology of cartilage, bone, and tendon. Birth Defects Research (Part C) 102:13–36, 2014. © 2014 Wiley Periodicals, Inc.     

ErbB4 signaling in the mammary gland is required for lobuloalveolar development and Stat5 activation during lactation.

Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4DeltaIC) as a transgene in the mammary gland. Despite the expression of ErbB4DeltaIC from puberty through later stages of mammary development, an ErbB4DeltaIC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4DeltaIC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, beta-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and alpha-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4DeltaIC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.     

Overexpression of ErbB2 receptor inhibits IGF-I-induced Shc-MAPK signaling pathway in breast cancer cells

Overexpression of the ErbB2 receptor in one-third of human breast cancers contributes to the transformation of epithelial cells and predicts poor prognosis for breast cancer patients. We report that the overexpression of ErbB2 inhibits IGF-I-induced MAPK signaling. IGF-I-induced MAPK phosphorylation and MAPK kinase activity are reduced in ErbB2 overexpressing MCF-7/HER2-18 cells relative to control MCF-7/neo cells. In SKBR3/IGF-IR cells, reduction of ErbB2 by antisense methodology restores the IGF-I-induced MAPK activation. The inhibition of IGF-I-induced MAP kinase activation in ErbB2 overexpressing breast cancer cells is correlated with decreased IGF-I-induced Shc tyrosine-phosphorylation, leading to a decreased association of Grb2 with Shc and decreased Raf phosphorylation. However, IGF-I-induced tyrosine-phosphorylation of IGF-I receptor and IRS-I and AKT phosphorylation were unaffected by ErbB2 overexpression. Consistent with these results, we observed that the proportion of IGF-I-stimulated proliferation blocked by the MAPK inhibitor PD98059 fell from 82.6% in MCF-7/neo cells to 41.2% in MCF-7/HER2-18 cells. These data provide evidence for interplay between the IGF-IR and ErbB2 signaling pathways. They are consistent with the view that the IGF-IR mediated attenuation of trastuzumab-induced growth inhibition we recently described is dependent on IGF-I-induced PI3K signaling rather than IGF-I-induced MAPK signaling.     

Expression of the c-fos proto-oncogene in bone, cartilage and tooth forming tissues during mouse development

The c-fos proto-oncogene is the cellular homologue of v-fos identified as the bone transforming gene of the FBJ and the FBR murine osteosarcoma viruses. We show here, using a sensitive in situ hybridization method, that the c-fos proto-oncogene is expressed in the cartilage, bone and tooth forming tissues during mouse development. This result suggests that the tumors observed after infection by the FBJ viral complex and c-fos overexpression in transgenic mice occur in those tissues in which c-fos is expressed during development.     

Stimulated ErbB4 internalization is necessary for neuregulin signaling in neurons

Neuregulin-1 (NRG1) plays an important role in neural development, synapse formation, and synaptic plasticity by activating ErbB receptor tyrosine kinases. Although ligand-induced endocytosis has been shown to be important for many receptor tyrosine kinases, whether NRG1 signaling depends on ErbB endocytosis remains controversial. Here, we provide evidence that ErbB4, a prominent ErbB protein in the brain, becomes internalized in NRG1-stimulated neurons. The induced ErbB4 endocytosis requires its kinase activity. Remarkably, inhibition of ErbB endocytosis attenuates NRG1-induced activation of Erk and Akt in neurons. These observations indicate a role of ErbB endocytosis in NRG1 signaling in neurons.     

Requirement of fibroblast growth factor signaling for regeneration of epiphyseal morphology in rabbit full-thickness defects of articular cartilage

The involvement of fibroblast growth factor-2 (FGF-2) during the repair process in rabbit full-thickness defects of articular cartilage was studied. Fibroblast growth factor-2 (50 pg/h) was administered for 2 weeks in a 5mm defect of articular cartilage, which is large enough not to repair spontaneously. The administration of FGF-2 resulted in the regeneration of the articular cartilage and the subchondral bone within 8 weeks. In these defects, undifferentiated mesenchymal cells initiated chondrogenic differentiation coupled with replacement by subchondral bone, resulting in the resurfacing of the defects with hyaline cartilage and the recovery of subchondral bone up to the original bone–articular cartilage junction. In rabbits, full-thickness defects are capable of regenerating articular cartilage as long as the defect size is limited to ≤3 mm in diameter. In the defects, strong immunoreactivity for FGF-2 was observed in the granulation tissue filling the defects in the early stage of repair, in association with the expression of FGF-2 mRNA shown by in situ hybridization. Once the undifferentiated mesenchymal cells had differentiated into chondrocytes, both the immunoreactivity and the in situ hybridization signal declined significantly. Upon the local administration of a monoclonal antibody against FGF-2 (bFM-1, 50ng/h), the defects were filled with fibrous tissue and no resurfacing hyaline cartilage was formed. Compared to the non-treated defects, there were marked increases in FGF-2 immunoreactivity and the overexpression of FGF-2 mRNA in the reparative tissue in the bFM-1 -treated defects. This rebound phenomenon indicates that the autocrine FGF-2 signaling is critically important for the regeneration of articular cartilage.     

PTPN21 exerts pro-neuronal survival and neuritic elongation via ErbB4/NRG3 signaling

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene–gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919 bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21–NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.     

Muc4 is required for activation of ErbB2 in signet ring carcinoma cell lines

Signet-ring cell carcinoma is one of the most malignant tumors, classified histologically as a poorly differentiated adenocarcinoma. The ErbB2/ErbB3 complex is often constitutively activated, which suggests that the ErbB2/ErbB3 signaling pathway may be important for malignancy of this tumor. However, the mechanism underlying this activation has not been understood. Here, we show that ErbB2 and Muc4 bind in signet ring carcinoma cells, which was not seen in highly differentiated adenocarcinoma cell lines. ErbB3 was suggested to be a substrate of ErbB2 because knockdown of ErbB2 resulted in less phosphorylation of ErbB3. Inhibition of expression of Muc4 at the cell surface by the treatment of the cells with benzyl-GalNac, an inhibitor of mucin secretion, blocked phosphorylation of ErbB3, suggesting that activity of ErbB2 depends on the expression of Muc4. These results supply the biochemical backgrounds in recent studies suggesting the contribution of Muc4 in the tumorigenesis.     

Effect of exercise on bone and articular cartilage in heterozygous manganese superoxide dismutase (SOD2) deficient mice

Abstract

Reactive oxygen species (ROS) are involved in both bone and cartilage physiology and play an important role in the pathogenesis of osteoporosis and osteoarthritis. The present study investigated the effect of running exercise on bone and cartilage in heterozygous manganese superoxide dismutase (SOD2)-deficient mice. It was hypothesized that exercise might induce an increased production of ROS in these tissues. Heterozygous SOD2-deficient mice should exhibit an impaired capability to compensate, resulting in an increased oxidative stress in cartilage and bone. Thirteen female wild type and 20 SOD2^+/? mice (aged 16 weeks) were randomly assigned to a non-active wild type (SOD2^+/+Con, n = 7), a trained wild type (SOD2^+/+Run, n = 6), a non-active SOD2^+/? (SOD2^+/?Con, n = 9) and a trained SOD2^+/? (SOD2^+/?Run, n = 11) group. Training groups underwent running exercise on a treadmill for 8 weeks. In SOD2^+/? mice elevated levels of 15-F_2t-isoprostane and nitrotyrosine were detected in bone and articular cartilage compared to wild type littermates. In osteocytes the elevated levels of these molecules were found to be reduced after exercise while in chondrocytes they were increased by aerobic running exercise. The observed changes in oxidative and nitrosative stress did neither affect morphological, structural nor mechanical properties of both tissues. These results demonstrate that exercise might protect bone against oxidative stress in heterozygous SOD2-deficient mice.