Maria E. Carvajal

AN INTRAMEMBRANE MODULATOR OF THE ERBB2 RECEPTOR TYROSINE KINASE THAT POTENTIATES NEUREGULIN SIGNALING

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.

Expression of the receptor tyrosine kinases, epidermal growth factor receptor, ErbB2, and ErbB3, in human ocular surface epithelia.

Purpose. To investigate the distribution and relative level of expression of the receptor tyrosine kinases, epidermal growth factor receptor (EGFR), ErbB2 and ErbB3, in human ocular surface epithelia. Methods. Immunofluorescent staining was performed to identify expression of the EGFR, ErbB2 and ErbB3 in the corneal, limbal and conjunctival epithelium in tissue sections and impression cytologies taken from normal human eyes. Western blotting was undertaken to confirm the results of immunofluorescent staining. Results. The three receptor tyrosine kinases, EGFR, ErbB2 and ErbB3, were detected in human corneal, limbal and conjunctival epithelia by immunofluorescent staining. Strong staining for the EGFR was observed in the basal epithelial cells at all 3 sites and throughout the corneal epithelium. Minimal or no staining for the EGFR was observed in the superficial conjunctival and limbal epithelia. The strongest staining for ErbB2 and ErbB3 was observed in the superficial ocular surface epithelium. All three receptors were detected in the corneal, limbal and conjunctival epithelium by western blot. Conclusion. EGFR, ErbB2 and ErbB3 are expressed by the ocular surface epithelia. EGFR is preferentially expressed by the basal epithelial cells that have the greatest proliferative potential. In contrast, ErbB2 and ErbB3 are preferentially expressed by the superficial differentiated ocular surface epithelia.

ErbB2 and Its Ligand Muc4 (Sialomucin Complex) in Rat Lacrimal Gland

The lacrimal gland is responsible for production of many of the major components of the ocular tear fluid. Mucins are important components of the tear film, but the cellular source of these mucins has been uncertain.1 Recently, we showed one of the components produced by the rat lacrimal gland is the mucin Muc4,2 also known as sialomucin complex (SMC). The lacrimal gland produces soluble and membrane forms of Muc4/SMC. The soluble form may contribute to the ocular tear film, but that raises the question of the role of the membrane form in the gland. One of the intriguing aspects of Muc4/SMC is that it has two epidermal growth factor (EGF)-like domains and can act as an intramembrane ligand for the receptor tyrosine kinase ErbB2.3 These characteristics encouraged us to analyze the expression of ErbB2 in the lacrimal gland and determine if an ErbB2-Muc4/SMC complex is present.

Association of the Ras to Mitogen-activated Protein Kinase Signal Transduction Pathway with Microfilaments EVIDENCE FOR A p185 neu -CONTAINING CELL SURFACE SIGNAL TRANSDUCTION PARTICLE LINKING THE MITOGENIC PATHWAY TO A MEMBRANE-MICROFILAMENT ASSOCIATION SITE

Microvilli of the aggressive 13762 ascites mammary adenocarcinoma contain a large, microfilament-associated signal transduction particle whose scaffolding is a stable glycoprotein complex (Li, Y., Hua, F., Carraway, K. L., and Carraway, C. A. C. (1999) J. Biol. Chem. 274, 25651–25658) associated with the growth factor receptor p185 neu . The receptor is constitutively tyrosine-phosphorylated in the cells and microvilli, predicting that it should recruit mitogenic pathway components to this membrane-microfilament interaction site. Immunoprecipitation of cell lysates with anti-phosphotyrosine and immunoblotting showed phosphorylated forms of the mitogenic pathway proteins Shc and MAPK in addition to p185 neu , suggesting that the Ras to MAPK mitogenic pathway is activated. Immunoblotting of p185 neu -containing microvillar fractions revealed the presence in each of stably associated Shc, Grb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase, as well as the transcription factor-phosphorylating kinase Rsk. All of these pathway components co-immunoprecipitated with p185 neu from cleared lysates of microvilli solubilized under microfilament-depolymerizing conditions. The recruitment of constitutively phosphorylated p185 neu and the activated mitogenic pathway proteins to this membrane-microfilament interaction site provides a physical model for integrating the assembly of the mitogenic pathway with the transmission of growth factor signal to the cytoskeleton. This linkage is probably a requisite step in the global cytoskeleton remodeling accompanying mitogenesis.

Increased expression of the type 1 growth factor receptor family in the conjunctival epithelium of patients with keratoconjunctivitis sicca.

PURPOSE To investigate the expression of type 1 growth factor receptors (epidermal growth factor receptor, ErbB2, and ErbB3) in the conjunctival epithelium of patients with keratoconjunctivitis sicca. METHODS Immunofluorescent staining and Western blotting were performed to grade the level of expression of the epidermal growth factor receptor ErbB2, and ErbB3 in conjunctival epithelial impression cytologies taken from both eyes of seven normal subjects and 22 patients with keratoconjunctivitis sicca. RESULTS Epidermal growth factor receptor staining was observed in a greater percentage of keratoconjunctivitis sicca than normal samples (P <.05). ErbB2 and ErB3 staining in the apical conjunctival epithelium was observed in both groups, but stronger ErbB2 and ErbB3 staining was noted in keratoconjunctivitis sicca conjunctival samples (P <.05). The relative levels of expression of these receptor proteins on immunoblots were consistent with immunofluorescent staining. On immunoblots, epidermal growth factor receptor protein was detected in 50% of keratoconjunctivitis sicca samples, but none of the normal samples (P <.025). The expression of ErbB2 and ErbB3 on immunoblots was also greater in the keratoconjunctivitis sicca samples (P <.05). Immunofluorescent staining scores for these receptors were correlated with conjunctival lissamine green staining scores (r =. 574, P <.01 for epidermal growth factor receptor; r =.620, P <.0025 for ErbB2; r =.502, P <.025 for ErbB3) and with corneal fluorescein staining (r =.409, P <.05 for ErbB2; r =.588, P <.005 for ErbB3). CONCLUSION The expression of the type 1 growth factor receptors is significantly greater in the conjunctival epithelium of eyes with keratoconjunctivitis sicca than normal eyes. The increased expression of these receptors was positively correlated with ocular surface dye staining. The increased expression of these receptors may contribute to the abnormal growth and differentiation of the conjunctival epithelium that occurs in keratoconjunctivitis sicca.

MUC4 (Sialomucin Complex) Expression in Salivary Gland Tumors and Squamous Cell Carcinoma of the Upper Aerodigestive Tract

OBJECTIVES: This study investigates MUC4 expression in normal squamous epithelia and squamous cell carcinoma (SCC) of the upper aerodigestive tract (UADT), and in salivary gland neoplasms. STUDY DESIGN: MUC4 antigens in tumor and adjacent normal tissue are localized by immunocytochemical studies. Fresh frozen tissues from surgical resection specimens are further analyzed by Western blot. RESULTS: MUC4 is identified by immunocytochemical staining throughout the normal UADT mucosa, in 34 of 40 primary UADT SCC, and in 11 of 12 metastatic cervical lymph nodes. A trend toward decreased MUC4 staining in moderately and poorly differentiated tumors is noted. Immunoblots show MUC4 in 4 of 5 SCC analyzed. Immunocytochemical staining of MUC4 in 13 major and minor salivary gland neoplasms reveal variable staining of normal and neoplastic tissue. MUC4 is demonstrated in immunoblots of normal parotid tissue and in the single parotid malignancy analyzed, but is not demonstrated in one minor salivary gland malignancy. These findings characterize normal UADT mucosal and salivary MUC4 expression, and MUC4 expression in SCC of the UADT and in salivary gland tumors. SIGNIFICANCE: Correlation of MUC4 expression with clinical outcomes may establish MUC4 as a potential molecular prognostic marker for these tumors.

Sialomucin complex (rMuc4) expression during development of the rat cornea.

Purpose. To determine changes in sialomucin complex (SMC, rat Muc4) expression in rat ocular surface tissue during development from birth to adult. Methods. Immunoblot and immunocytochemical analyses were performed on isolated corneal tissue and rat eyes, respectively, from animals at days 1, 9 and 15 after birth. Adult animals were used for comparison. Results. SMC was detected by immunoblotting at all ages (day 1-adult) and increased into adulthood. Immunocytochemical localization also showed steadily increasing amounts of SMC to adulthood. The SMC was located at the apical surface of the corneal epithelium and in the superficial layers of the stratified corneal epithelium. Conclusions. SMC at the corneal surfaces steadily increases during development to adulthood, but does not exhibit a marked transition in expression at the time of eye-opening.

ERBB2 AND ITS LIGAND MUC4 (SIALOMUCIN COMPLEX) IN RAT LACRIMAL GLAND.

The lacrimal gland is an important source of components for the ocular tear fluid. Though mucins are not generally considered a product of the lacrimal gland, our results clearly show Muc4/SMC is produced by the gland in soluble and membrane forms. The secreted, soluble form is likely produced for the soluble phase of the ocular tear film. Analyses of ErbB2 and the Muc4/SMC-ErbB2 complex in the lacrimal gland suggest a second function for Muc4/SMC, a role in cell regulation through ErbB signaling. The nature of those signals and the cell functions they regulate will be subjects for future investigations.