MUC1 Initiates a Calcium Signal after Ligation by Intercellular Adhesion Molecule-1

Abstract

The MUC1 mucin is normally restricted to the apical surface of breast epithelial cells. In tumors, it is frequently overexpressed and underglycosylated. The MUC1 peptide core mediates firm adhesion of tumor cells to adjacent cells via binding to intercellular adhesion molecule-1 (ICAM-1). There is increasing evidence that MUC1 is involved in signaling, with current reports focusing on phosphorylation of the MUC1 cytoplasmic tail after indirect or artificial modes of stimulation. ICAM-1 is the only known direct ligand of the MUC1 extracellular domain. The data presented herein show that MUC1 expressed on the surface of breast cancer cell lines or transfected 293T cells can initiate a calcium-based oscillatory signal on contact with ICAM-1-transfected NIH 3T3 cells, and we present a novel method of quantifying and comparing calcium oscillations. The MUC1-induced signal appears to be distinct from those previously described, and may involve a Src family kinase, phosphoinositol 3-kinase, phospholipase C, and lipid rafts, but not mitogenactivated protein kinase. As calcium signaling has been associated with cytoskeletal change and motility, it is possible that the functions of MUC1 include heterotypic cell-cell adhesion followed by a calcium-based promigratory signal within tumor cells, thus facilitating metastasis.The MUC1 mucin has been well established as a tumor marker in breast cancer and is implicated in metastatic spread (1). Phosphorylation of the MUC1 cytoplasmic tail (2-4) allows MUC1 to associate with potential oncogenes (5-8). MUC1 can also be indirectly stimulated to initiate signaling cascades, through epidermal growth factor receptor (EGFR) 1 (9, 10) or antibodies directed against CD8-MUC1 chimeras (11, 12), resulting in activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) pathway. Our laboratory has previously identified intercellular adhesion molecule-1 (ICAM-1) as a natural, endogenous ligand for MUC1 (13,14). ICAM-1 is currently the only reported direct ligand for the MUC1 extracellular domain, and binding promotes adhesion of tumor cells to a simulated vessel wall construct under fluid flow conditions (15). In this report, we demonstrate a novel signaling paradigm, in which MUC1 participates in initiating intracellular calcium oscillations after direct stimulation by ICAM-1. As calcium signaling has previously been implicated in cytoskeletal remodeling and motility (16), we hypothesize that this signal is involved in tumor cell migration. Thus, the MUC1/ICAM-1 interaction may facilitate extravasation of tumor cells after mediating binding to the blood vessel wall. EXPERIMENTAL PROCEDURESReagents-The CT2 antibody against the MUC1 cytoplasmic domain and the pC1Neo TRϩ FLAG plasmid carrying the MUC1 gene were generously provided by Dr. Sandra Gendler, Mayo Clinic, Scottsdale, AZ. The MUC1 gene was PCR amplified from the plasmid and inserted into the Clontech pEYFP-N1 plasmid at the BsrGI/NotI cut sites. A synthetic MUC1-specific signal sequence, TCGACTAG...

MUC1 Initiates Src-CrkL-Rac1/Cdc42–Mediated Actin Cytoskeletal Protrusive Motility after Ligating Intercellular Adhesion Molecule-1

“…This indicates that MUC1, although without intrinsic kinase activity (19), could function as a scaffold protein in signal transduction. Supporting this, we have shown that MUC1 can initiate an intracellular calcium signal in response to ICAM-1 ligation (20). Calcium-based signaling is frequently implicated in actin cytoskeletal ''treadmilling'' dynamics and cell migration (21).…”

“…The adaptor protein CrkL, as a downstream mediator of Src, plays a crucial role in the spatial-temporal regulation of Rho GTPase Rac1-Cdc42 activation and cell migration (30). Our previous work has shown that Src family kinase and PLC are involved in the MUC1/ICAM-1 interaction -initiated calcium signal (20). These findings suggest that CrkL may cooperate with Src in this MUC1/ICAM-1 interaction -induced actin cytoskeletal protrusive motility.…”

“…Calcium-based signaling is frequently implicated in actin cytoskeletal ''treadmilling'' dynamics and cell migration (21). Also, the signal mediators Src family kinase and PLCg involved in the MUC1/ ICAM-1 -induced calcium oscillation (20) could play crucial roles in directing cell migration (22,23). All these findings imply that, in addition to mediating cell adhesion, the MUC1/ ICAM-1 interaction may play a signaling role in cell motility.…”

“…The MUC1-transfected 293T SYM3, SYM25, and SYM33 sublines, as previously described (20), were maintained in DMEM containing 10% FBS and 200 Ag/mL G418. Human ICAM-1 -transfected NIH3T3 cells and the mock-transfected counterparts were a generous gift of Dr. Ken Dimock (University of Ottawa, Ottawa, ON, Canada) and were maintained in DMEM containing 10% FBS and 2 Ag/mL blasticidin S. To generate the Flp-In T-REx 293 MUC1-inducible expression system, the FlpIn T-REx 293 parental cells (Invitrogen) were cotransfected with pcDNA5/FRT/TO-MUC1 plasmids and pOG44 plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol.…”

The cell surface mucin MUC1 limits the severity of influenza A virus infection

“…6 These features enable cs-mucins to provide a barrier function to mucosal pathogens by displaying sites for pathogen binding, acting as a releasable decoy, and sterically blocking binding of pathogens to underlying cellular receptors. The cytoplasmic tail domain of cs-mucins is highly conserved across species, undergoes both serine and tyrosine phosphorylation, and interacts with kinases and adaptor molecules, [7][8][9][10][11] consistent with a role in signal transduction in response to pathogen binding. As IAV specifically binds sialic acid-expressing receptors to trigger its entry by endocytosis into epithelial cells, the virus also has the potential to interact with sialic acid on cs-mucins, which may modulate both the infection process and the host response.…”