Progress 10/01/02 to 09/30/08
Outputs
OUTPUTS: 1. Skeletal myoblasts (muscle cells) grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated "reserve cells". We found that the expression level of metalloprotease disintegrin ADAM12 is much higher in proliferating myoblasts and in reserve cells than in myotubes and that the presence of ADAM12 plays an important role in determination of the pool of reserve cells. 2. We described a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulated that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. 3. We produced individual domains of ADAM12 protein in insect cells, purified these recombinant proteins and characterized them using functional assays and biophysical methods. We found that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich domains differ from the structures present in the intact ADAM12. 4. Notch receptors participate in key aspects of organogenesis in the developing embryo, maintain stem cells viability in the adult, and have been implicated in multiple human cancers. Delta-like 1 (Dll1) is a ligand (binding partner) for Notch receptors. We demonstrated that Dll1 is cleaved by ADAM12 and by several other members of the ADAM family of proteases. We showed that Dll1 cleavage by ADAM12 increases Notch signaling in neighboring cells and increases Notch activity in the same cell. 5. We showed that the cleavage of Dll1 by ADAMs is a key step in muscle cell differentiation and self-renewal. Using mouse primary myoblasts cultured in vitro or C2C12 myogenic cells, we found that Notch activity is essential for maintaining the expression of Pax7, a transcription factor associated with self-renewal. Dll1 is shed by ADAMs in a pool of Pax7-positive C2C12 reserve cells, but it remains intact in differentiated myotubes. Inhibition of Dll1 cleavage by a soluble, dominant-negative mutant form of ADAM12 leads to elevation of Notch signaling, inhibition of differentiation, and expansion of the pool of self-renewing Pax7-positive cells. We concluded that that the proteolytic processing of Dll1 helps achieve an asymmetry in Notch signaling in initially equivalent myogenic cells and helps sustain the balance between differentiation and self-renewal. 6. We showed that two breast cancer-associated mutations in ADAM12 inhibit the maturation and activation of ADAM12. Using a panel of normal and cancer cells, we demonstrated that ADAM12 mutants are retained in the endoplasmic reticulum and are not transported to the cell surface. Our results suggest that the breast cancer-associated mutations interfere with the intracellular trafficking of ADAM12 and result in loss of the functional ADAM12 at the cell surface. PARTICIPANTS: Individuals: Anna Zolkiewska, Ph.D., Principal Investigator; Hui Li, Ph.D., Research Associate (post-doctoral fellow); Emilia Syta, Research Assistant (technician); Joanna Gruszczynska-Biegala (technician); Danqiong Sun, Graduate Student; Emilia Dyczynska, Graduate Student; Yi Cao, Graduate Student; Zhefeng Zhao, Graduate Student; Haiqing Yi, Graduate Student; Michael Asiedu, Graduate Student; An Zhang, Graduate Student; Dan Liu, Graduate Student Partner Organizations: Kansas State University Training or professional development: Hui Li, Ph.D., Research Associate (post-doctoral fellow); Danqiong Sun, Graduate Student; Emilia Dyczynska, Graduate Student; Yi Cao, Graduate Student; Zhefeng Zhao, Graduate Student; Haiqing Yi, Graduate Student; Michael Asiedu, Graduate Student; An Zhang, Graduate Student; Dan Liu, Graduate Student TARGET AUDIENCES: Scientists working in the area of tumor biology, muscle biology, and muscle disease Cancer patients and patients suffering from muscle diseases (such as muscular dystrophies and muscle fibrosis) Animal health professionals involved in treatment of cancer and muscle disease PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our studies may help develop therapeutic interventions in breast cancer and in muscle diseases based on functionality of ADAM12 and/or related signaling pathways. Our studies have, therefore, important implications in human and veterinary medicine. The results of our studies may increase life expectancy of affected individuals and therefore they may improve the overall quality of life of families and communities.
Publications
- Dyczynska, E., Syta, E., Sun, D., and Zolkiewska, A. (2008) Breast cancer-associated mutations in metalloprotease disintegrin ADAM12 interfere with the intracellular trafficking and processing of the protein. Int. J. Cancer 122, 2634-2640
- Zolkiewska, A. (2008) ADAM proteases: ligand processing and modulation of the Notch pathway. Cel. Mol. Life Sci. 65, 2056-2068
- Sun, D., Li, H., and Zolkiewska, A. (2008) The role of Delta-like 1 shedding in muscle cell self-renewal and differentiation. J. Cell Sci., 121, 3815-3823
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: A. ADAM12 has recently emerged as a Candidate Cancer Gene in a comprehensive genetic analysis of human breast cancers. Three somatic mutations in ADAM12 were observed at significant frequencies in breast cancers: D301H, G479E, and L792F. The first two of these mutations involve highly conserved residues in ADAM12 and our computational sequence analysis confirmed that they might be cancer-related. We showed that the corresponding mutations in mouse ADAM12 inhibit the proteolytic processing and activation of ADAM12 in NIH3T3, COS-7, CHO-K1 cells and in MCF-7 breast cancer cells. The D/H and G/E ADAM12 mutants exert a dominant-negative effect on the processing of the wild-type ADAM12. Immunofluorescence analysis and cell surface biotinylation experiments demonstrate that the D/H and G/E mutants are retained inside the cell and are not transported to the cell surface. Consequently, the D/H and G/E mutants, unlike the wild-type ADAM12, are not capable of shedding Delta-like l, a
ligand for Notch receptor, at the cell surface, or of stimulating cell migration. Our results suggest that the breast cancer-associated mutations interfere with the intracellular trafficking of ADAM12 and result in loss of the functional ADAM12 at the cell surface. B. In a different line of studies, we examined the mechanisms regulating ADAM12 expression in mammalian cells. We showed that treatment of cultured cells with transforming growth factor beta, TGF-beta, leads to increased levels of ADAM12 protein and mRNA. The increase of ADAM12 mRNA occurred at the transcriptional level, as it was blocked by actinomycin A, an inhibitor of transcription, and not by cyclohexamide, an inhibitor of translation. ADAM12 protein was also strongly up-regulated in cells expressing a constitutively active form of Notch receptor. These results provide a new insight into the regulation of ADAM12 expression that may be relevant to cancer progression and the function of muscle stem cells (see below). C.
During the last year, we have been continuing our studies on ADAM12-mediated proteolysis in muscle development and the function of muscle stem cells. We showed that ADAM12, together with several other ADAM proteases, cleaves Delta, a ligand for Notch receptor. ADAM12 thus modulates the Notch pathway, one of the major signaling pathways in muscle stem cells. Cleavage by ADAM12 leads to the shedding of the extracellular domain of Delta from the cell surface, down-regulation of the Notch signaling in the neighboring cells, and up-regulation of the Notch activity in the cell in which the shedding occurs. Cells with increased Notch signaling express Pax7, a transcription factor that is essential for the maintenance of muscle stem cells. We showed that when primary cultures of myogenic cells isolated from mouse skeletal muscle are treated with inhibitors of Delta cleavage, the formation of Pax7-positive cells is impaired. These results brought us to conclusion that ADAM-mediated shedding of
Delta represents a physiologically important mechanism of self-renewal of muscle stem cells.
PARTICIPANTS: Anna Zolkiewska-PI
TARGET AUDIENCES: Life scientists and educators
Impacts
Our studies may help develop therapeutic interventions in breast cancer and in muscle diseases based on functionality of ADAM12 and/or related signaling pathways. Our studies have, therefore, important implications in human and veterinary medicine.
Publications
- Dyczynska, E., Sun, D., Yi, H., Sehara-Fujisawa, A., Blobel, C. P., and Zolkiewska, A. (2007) Proteolytic Processing of Delta-like 1 by ADAM Proteases. J. Biol. Chem. 282, 436-444
- Dyczynska, E., Syta, E., Sun, D., and Zolkiewska, A. (2007) Breast Cancer-Associated Mutations in Metalloprotease Disintegrin ADAM12 Interfere with the Intracellular Trafficking and Processing of the Protein. Int. J. Cancer, in press
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Progress 01/01/06 to 12/31/06
Outputs
A. During the last year, we published our studies on cleavage of Delta-like 1 (Dll1) by ADAM12. Dll1 is a mammalian ligand for Notch receptors. Dll1 present at the surface of a signal sending cell binds to Notch at the surface of a signal receiving cell. Upon this binding, Notch signaling cascade is initiated in the receiving cell. It was shown previously that Dll1 undergoes proteolytic processing in its extracellular domain by ADAM10, a member of the ADAM family of proteins. In our work, we demonstrated that Dll1 represents a substrate for several other members of the ADAM family, including ADAM12. Our findings expand the role of ADAM proteins in the regulation of Notch signaling. B. We have been continuing our studies aimed at identification of cell surface proteins that interact with ADAM12. We produce the soluble, extracellular domain of mouse ADAM12 in Drosophila S2 cells and purify the recombinant protein from culture medium. We then add the purified protein to
mouse skeletal muscle cells grown in vitro. As published by our laboratory earlier, the soluble extracellular domain of ADAM12 inhibits muscle cell differentiation. During the last year, we gather the evidence that this recombinant ADAM12 domain exerts its function by binding to Delta-like 1 at the cell surface and by activating the Notch signaling, which is well known to interfere with muscle differentiation. C. We have initiated studies on the role of ADAM12 in cancer invasion and metastasis. Recently, ADAM12 has been associated with invasive breast and prostate cancers. While ADAM12 is not present in normal mammary or prostate epithelium, the level of ADAM12 expression rises dramatically in epithelium-derived carcinomas. Furthermore, several mutations in ADAM12 have been associated with increased risk of breast cancer. Mice with increased level of ADAM12 develop more aggressive breast tumors. Conversely, the lack of functional ADAM12 gene decreases tumor size and slows down tumor
invasion and metastasis. We have determined that tumor growth factor beta 1 (TGFbeta1) induces expression of ADAM12 in mouse mammary gland epithelial cell line NMuMG. TGFbeta1 is also known to promote epithelial to mesenchymal transformation in these cells and stimulate cell migration and invasiveness. We have begun to study whether the presence of ADAM12 is necessary for the increased cell migration and whether ADAM12 alone is sufficient to induce the mesenchymal phenotype in these cells.
Impacts
Our studies may help understand skeletal muscle regeneration and cancer progression. Our studies have, therefore, important implications in human and veterinary medicine. Our research may help develop therapeutic approaches to increase muscle growth and repair, as well as to inhibit cancer invasion and metastasis.
Publications
- Zolkiewska, A., Dyczynska E., Sun, D., Tasheva, E., and Yi. H. (2006) Proteolytic Processing of the Notch Ligand Dll1 by Muscle-Specific ADAMs. Conference on "Frontiers in Myogenesis", Callaway Gardens, GA, April 2006, Abstracts published by the Society for Muscle Biology, p. 21
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Progress 01/01/05 to 12/31/05
Outputs
A. During the last year, we published our studies on the cell adhesion mediated by ADAM12 protein. We found that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supported efficient adhesion and spreading of C2C12 muscle cells. This adhesion was not mediated by beta1 integrins or proteoglycans, was myoblast-specific, and required the presence of both the metalloprotease domain and the disintegrin/cysteine-rich domains of ADAM12. Analysis of the recombinant proteins by far-UV circular dichroism suggested that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich/EGF-like domains differed from the structures present in the intact extracellular domain. Furthermore, the intact extracellular domain, but not the metalloprotease or the disintegrin/cysteine-rich/EGF-like fragment alone, inhibited C2C12 myoblast fusion. B. We have initiated the studies on the proteolytic activity of
ADAM12. ADAM12 contains in its extracellular portion a metalloprotease domain. The catalytic activity of the ADAM12 metalloprotease depends on the presence of zinc ions. We have demonstrated that ADAM12 cleaves another cell surface protein called Delta1. Delta1 present in one muscle cell interacts with Notch receptor in the neighboring cell. This activates the Notch signaling pathway that leads to cell proliferation during muscle regeneration. However, when a sufficient number of new cells has been generated during the regeneration process, the Notch signaling needs to be turned off. ADAM12-mediated cleavage of Delta1 should result in such a termination of Notch signaling and should enable cells to proceed to myogenic differentiation. Thus, ADAM12-mediated cleavage of Delta1 may play a significant role during muscle regeneration. Furthermore, any de-regulation of ADAM12 observed in certain muscle diseases or during aging may lead to abnormal muscle cell proliferation. C. We have
published two papers related to the regulation of integrin alpha7, a cell adhesion molecule, by ADP-ribosylation. ADP-ribosylation is a post-translational modification of proteins catalyzed by extracellular enzymes called ecto-ADP-ribosyltransferases. These enzymes catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to arginine residues in cell surface proteins. Since under normal physiological conditions the concentration of extracellular NAD is very low but it raises significantly upon tissue injury or membrane stress, we have postulated that the main role of ecto-ARTs is to ADP-ribosylate and regulate the function of membrane receptors in response to elevated levels of NAD.
Impacts
Our studies may help understand the molecular steps that control the function of muscle satellite cells during repair after injury of muscle. Our studies have, therefore, important implications in human and veterinary medicine. Our research may help develop some therapeutic approaches to increase muscle growth, regeneration, and repair.
Publications
- Zhao, Z., Gruszczynska-Biegala, J., and Zolkiewska, A. 2005. ADP-ribosylation of integrin alpha7 modulates the binding of integrin alpha7 beta1 to laminin. Biochem. J. 385: 309-317.
- Yi, H., Gruszczynska-Biegala, J., Wood, D., Zhao, Z., and Zolkiewska, A. 2005. Cooperation of the metalloprotease, disintegrin, and cysteine-rich domains of ADAM12 during inhibition of myogenic differentiation. J. Biol. Chem. 280: 23475-23483.
- Zolkiewska, A. 2005. Ecto-ADP-ribose transferases: cell-surface response to local tissue injury. Physiology 20: 374-381.
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Progress 01/01/04 to 12/31/04
Outputs
During the last year, we continued our studies on the role of ADAM12 protein in muscle regeneration. We found that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supports efficient adhesion and spreading of C2C12 myoblasts. This adhesion is not mediated by beta1 integrins or proteoglycans, is myoblast-specific, and requires the presence of both the metalloprotease domain and the disintegrin/cysteine-rich domains of ADAM12. Analysis of the recombinant proteins by far-UV circular dichroism suggests that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich/EGF-like domains differ from the structures present in the intact extracellular domain. Furthermore, the intact extracellular domain, but not the metalloprotease or the disintegrin/cysteine-rich/EGF-like fragment alone, decreases the expression of cell cycle inhibitor p21 and myogenin, two markers of differentiation, and
inhibits C2C12 myoblast fusion. Thus, the novel protein-protein interaction discovered in our laboratory may have important biological consequences during myoblast differentiation. We have previously found that the extracellular domain of integrin alpha7, a muscle-specific cell adhesion molecule, is modified by the reaction called ADP-ribosylation. During the past year, we continued our work in this area. We studied the effect of ADP-ribosylation on the structure or function of integrin alpha7. We showed that ADP-ribosylation of integrin alpha7 takes place exclusively in differentiated muscle cells and that this post-translational modification modulates the affinity of alpha7beta1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa stalk region of alpha7 that takes place at micromolar NAD concentrations increases the binding of the alpha7beta1 dimer to laminin. Increased in vitro binding of integrin alpha7beta1 to laminin after ADP-ribosylation of the 37-kDa fragment of
alpha7 requires the presence of manganese ions and it is not observed in the presence of magnesium. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand binding site of alpha7 that occurs at higher NAD concentrations inhibits the binding of integrin alpha7beta1 to laminin. Furthermore, incubation of C2C12 muscle cells with NAD increases the expression of an epitope on integrin beta1 subunit recognized by monoclonal antibody 9EG7. We proposed a hypothesis that ADP-ribosylation represents a mechanism of regulation of integrin alpha7beta1 function in myofibers in vivo when the continuity of the membrane is compromised and NAD is available as a substrate for ADP-ribosylation outside the cells.
Impacts
Our studies may help understand the molecular steps that control the function of muscle satellite cells during repair after injury of muscle. Our studies have, therefore, important implications in human and veterinary medicine. Our research may help develop some therapeutic approaches to increase muscle growth, regeneration, and repair.
Publications
- Zhao, Z., Gruszczynska-Biegala, J., Cheuvront, T., Yi, H., von der Mark, H., von der Mark, K., Kaufman, S. J., and Zolkiewska, A. 2004. Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrinalpha7beta1. Exp. Cell Res. 298, 28-37
- Zhao Z, Gruszczynska-Biegala J, and Zolkiewska A. 2004. ADP-ribosylation of integrin alpha7 modulates the binding of integrin alpha7beta1 to laminin. Biochem J., in press
- Yi, H., Gruszczynska-Biegala, J., Wood, D., and Zolkiewska, A. 2004. Cooperation of the metalloprotease, disintegrin, and cysteine-rich domains of ADAM12 during inhibition of myogenic differentiation. J. Biol. Chem., submitted
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Progress 01/01/03 to 12/31/03
Outputs
A. We have further characterized the interaction between the cell adhesion domain (DC domain) of ADAM12 and integrin alpha7beta1. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for manganese than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase at Tyr397. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulated that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle
development, function, and disease. We also concluded that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses. B. We found that that the expression level of metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in quiescent reserve cells than in differentiated myotubes. Inhibition of ADAM12 expression by siRNA was accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin alpha7A isoform). Overexpression of ADAM12 under the conditions that promote cell cycle progression led to up-regulation of p130 and p27, cell cycle arrest, and down-regulation of MyoD. Thus, enhanced expression of ADAM12 induced a quiescence-like phenotype and it did not stimulate differentiation. We also showed
that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity, as well as the cytoplasmic domain of ADAM12, were required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain was not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of "reserve" cells during myoblast differentiation in vitro, which share several characteristics with muscle satellite cells in vivo.
Impacts
ADAMs, a family of cell surface proteins containing A Disintegrin And Metalloprotease domains, play important roles in many biological processes involving cell surface proteolysis, cell-cell, or cell-matrix interactions. The molecular mechanism of the action of ADAM proteins is not known. Our studies will help to plan some therapeutic approaches that target ADAM proteins in tissue growth, regeneration, and repair.
Publications
- Cao, Y., Zhao, Z., Gruszczynska-Biegala, J., and Zolkiewska, A. 2003. Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro. Mol. Cell. Biol. 23, 6725-6738
- Liu, Z., Zolkiewska, A., and Zolkiewski, M. 2003. Characterization of human torsinA and its dystonia-associated mutant form. Biochem. J. 374, 117-122
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Progress 10/01/02 to 12/31/02
Outputs
We have been studying the role of ADAM12 protein, a member of the family of membrane metalloprotease-disintegrins, in differentiation and regeneration of skeletal muscle tissue. We have achieved a significant progress in identification of a binding partner for the cell adhesion domain of ADAM12. Using antibodies specific for integrin alpha7 and integrin beta1, we have demonstrated that integrin alpha7beta1 binds to the disintegrin-like/cysteine-rich (DC)domain of ADAM12 in vitro. Cells with surface expression of integrin alpha7beta1, but not control cells, adhered to plastic wells coated with recombinant DC domain of ADAM12. Finally, cells expressing integrin alpha7beta1 showed increased binding to a monolayer of cells expressing ADAM12. Binding of integrin alpha7beta1 to ADAM12 is a novel finding that has not been reported in the literature. Integrin alpha7 plays an important role in muscle development and disease. Mice lacking the alpha7 gene develop a specific form
of muscular dystrophy and mutations in the alpha7 gene in humans cause congenital myopathies. Since ADAM12 has been implicated in muscle development and regeneration as well, we believe that interactions between ADAM12 and integrin alpha7beta1 may have physiological significance.
Impacts
ADAMs, a family of cell surface proteins containing A Disintegrin And Metalloprotease domains, play important roles in many biological processes involving cell surface proteolysis, cell-cell, or cell-matrix interactions. The molecular mechanism of the action of ADAM proteins is not known. Our studies will help to plan some therapeutic approaches that target ADAM proteins in tissue growth, regeneration, and repair.
Publications
- Cao, Y., Kang, Q., Zhao, Z., and Zolkiewska, A. 2002. Intracellular Processing of Metalloprotease Disintegrin ADAM12. J. Biol. Chem. 277: 26403-26411.
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