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MUC1* mediates the growth of human pluripotent stem cells.

Hikita ST, Kosik KS, Clegg DO, Bamdad C - PLoS ONE (2008)

Bottom Line: Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23.Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells".These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology and Engineering, University of California Santa Barbara, Santa Barbara, California, USA.

ABSTRACT
The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells". Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

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Schematic and antibody recognition of full-length MUC1 versus the membrane-bound cleavage product MUC1*.A. Full-length MUC1 protein (MUC1-FL) is comprised of a cytoplasmic tail (CT), a transmembrane domain (TM), a self-aggregation domain (SAD), and hundreds of tandem repeats (TRs). B. Cleavage product, MUC1*, consists of the cytoplasmic tail, transmembrane domain, and at least 45 amino acids of the extracellular domain (ECD). Although the exact site(s) of cleavage remain somewhat uncertain, to our knowledge, no cleavage sites have been reported that leave less than a 45 amino acid ECD. Binding sites for antibodies VU4H5 and Anti-MUC1* are marked.
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pone-0003312-g001: Schematic and antibody recognition of full-length MUC1 versus the membrane-bound cleavage product MUC1*.A. Full-length MUC1 protein (MUC1-FL) is comprised of a cytoplasmic tail (CT), a transmembrane domain (TM), a self-aggregation domain (SAD), and hundreds of tandem repeats (TRs). B. Cleavage product, MUC1*, consists of the cytoplasmic tail, transmembrane domain, and at least 45 amino acids of the extracellular domain (ECD). Although the exact site(s) of cleavage remain somewhat uncertain, to our knowledge, no cleavage sites have been reported that leave less than a 45 amino acid ECD. Binding sites for antibodies VU4H5 and Anti-MUC1* are marked.

Mentions: We used three antibodies to probe the expression of MUC1 on hESCs: two that recognize the full-length protein (MUC1-FL) and one that recognizes the cleavage product, MUC1*. Both VU4H5 and HMPV are commercially available antibodies that bind to epitopes in the tandem repeats of the full-length protein (Fig. 1A). VU4H5 preferentially binds to underglycosylated MUC1, while HMPV recognizes full-length MUC1 in a glycosylation-independent manner and can bind to the fully glycosylated protein. Anti-MUC1* is a rabbit polyclonal antibody that was raised against a synthetic peptide that corresponds to the first forty-five (45) membrane-proximal amino acids of the extracellular domain, which comprises most if not all of the extracellular domain of the cleavage product, MUC1* (Fig. 1B). As we previously reported [1], although the epitope for Anti-MUC1* is present in the full-length protein, Anti-MUC1* does not bind to MUC1-FL when analyzed by Western blot or immunocytochemistry. Immunoprecipitation experiments show that Anti-MUC1* reacts very weakly with MUC1-FL. One possible explanation is that full-length MUC1 contains a self-aggregation domain that likely contributes to the protein's characteristic clustering and could sterically hinder the binding of ligands to the adjacent region which is the Anti-MUC1* epitope. Cleavage of MUC1 on cancer cells releases the bulk of the extracellular domain, including most if not all of the self-aggregation domain. It is not known whether or not Anti-MUC1* binds to alternative splice isoforms, such as MUC1/X, MUC1/Y or MUC1/Z [19], [20], which, like MUC1*, contain the Anti-MUC1* epitope and lack the tandem repeat region; one way that they differ from MUC1* is that their extracellular domains are more than one hundred (100) amino acids longer and contain the self-aggregation domain. Although MUC1/Y is not cleaved, it has been reported that MUC1/X/Z can be cleaved to yield a membrane-attached fragment that is essentially indistinguishable from the MUC1-FL cleavage product [21], and thus would likely be recognized by Anti-MUC1*.


MUC1* mediates the growth of human pluripotent stem cells.

Hikita ST, Kosik KS, Clegg DO, Bamdad C - PLoS ONE (2008)

Schematic and antibody recognition of full-length MUC1 versus the membrane-bound cleavage product MUC1*.A. Full-length MUC1 protein (MUC1-FL) is comprised of a cytoplasmic tail (CT), a transmembrane domain (TM), a self-aggregation domain (SAD), and hundreds of tandem repeats (TRs). B. Cleavage product, MUC1*, consists of the cytoplasmic tail, transmembrane domain, and at least 45 amino acids of the extracellular domain (ECD). Although the exact site(s) of cleavage remain somewhat uncertain, to our knowledge, no cleavage sites have been reported that leave less than a 45 amino acid ECD. Binding sites for antibodies VU4H5 and Anti-MUC1* are marked.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553196&req=5

pone-0003312-g001: Schematic and antibody recognition of full-length MUC1 versus the membrane-bound cleavage product MUC1*.A. Full-length MUC1 protein (MUC1-FL) is comprised of a cytoplasmic tail (CT), a transmembrane domain (TM), a self-aggregation domain (SAD), and hundreds of tandem repeats (TRs). B. Cleavage product, MUC1*, consists of the cytoplasmic tail, transmembrane domain, and at least 45 amino acids of the extracellular domain (ECD). Although the exact site(s) of cleavage remain somewhat uncertain, to our knowledge, no cleavage sites have been reported that leave less than a 45 amino acid ECD. Binding sites for antibodies VU4H5 and Anti-MUC1* are marked.
Mentions: We used three antibodies to probe the expression of MUC1 on hESCs: two that recognize the full-length protein (MUC1-FL) and one that recognizes the cleavage product, MUC1*. Both VU4H5 and HMPV are commercially available antibodies that bind to epitopes in the tandem repeats of the full-length protein (Fig. 1A). VU4H5 preferentially binds to underglycosylated MUC1, while HMPV recognizes full-length MUC1 in a glycosylation-independent manner and can bind to the fully glycosylated protein. Anti-MUC1* is a rabbit polyclonal antibody that was raised against a synthetic peptide that corresponds to the first forty-five (45) membrane-proximal amino acids of the extracellular domain, which comprises most if not all of the extracellular domain of the cleavage product, MUC1* (Fig. 1B). As we previously reported [1], although the epitope for Anti-MUC1* is present in the full-length protein, Anti-MUC1* does not bind to MUC1-FL when analyzed by Western blot or immunocytochemistry. Immunoprecipitation experiments show that Anti-MUC1* reacts very weakly with MUC1-FL. One possible explanation is that full-length MUC1 contains a self-aggregation domain that likely contributes to the protein's characteristic clustering and could sterically hinder the binding of ligands to the adjacent region which is the Anti-MUC1* epitope. Cleavage of MUC1 on cancer cells releases the bulk of the extracellular domain, including most if not all of the self-aggregation domain. It is not known whether or not Anti-MUC1* binds to alternative splice isoforms, such as MUC1/X, MUC1/Y or MUC1/Z [19], [20], which, like MUC1*, contain the Anti-MUC1* epitope and lack the tandem repeat region; one way that they differ from MUC1* is that their extracellular domains are more than one hundred (100) amino acids longer and contain the self-aggregation domain. Although MUC1/Y is not cleaved, it has been reported that MUC1/X/Z can be cleaved to yield a membrane-attached fragment that is essentially indistinguishable from the MUC1-FL cleavage product [21], and thus would likely be recognized by Anti-MUC1*.

Bottom Line: Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23.Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells".These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology and Engineering, University of California Santa Barbara, Santa Barbara, California, USA.

ABSTRACT
The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells". Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

Show MeSH
Related in: MedlinePlus