Here we have investigated the ability of laminin-1 and specific laminin-1-derived synthetic peptides to stimulate neuronal cell matrix metalloproteinase secretion. Zymographic analysis of conditioned media from laminin-1-treated PC12 and NG108-15 cells revealed a 72-kDa matrix metalloproteinase which was not secreted by untreated cells. Laminin-1 alpha1 chain-derived synthetic peptides, AASIKVAVSADR (LAM-L) and RKRLQVQLSIRT (AG-73), also stimulated PC12 cell secretion of a 72-kDa matrix metalloproteinase. We further investigated the structural requirements of AG-73 for cell attachment, neurite outgrowth, and matrix metalloproteinase secretion using a series of AG-73 analogs that had single amino acids substituted with alanine. At the substrate levels tested, the AG-73 peptide promoted the adhesion of 67% of the PC12 cells and neurite outgrowth in 71% of the PC12 cells. Substitutions in any one of the amino acids within the central LQVQ sequence resulted in a large reduction in cell attachment whereas substitution in the carboxyl terminal proximal amino acids L, S, and R had little effect on attachment. Alanine substitution of any of the amino terminal proximal LQV amino acids and the carboxyl terminal L, I, and R residues resulted in a 65-91% reduction in neurite outgrowth. These data demonstrate that the sequence requirements for cell attachment and neurite outgrowth were not necessarily coupled but that the sequence requirements for neurite outgrowth and matrix metalloproteinase secretion were identical. We conclude that laminin-1 is able to stimulate neuronal cells to secrete a matrix metalloproteinase. Further, this study identifies the LQVXLXIR laminin-1 alpha1 globular domain peptide to be capable of stimulating both neurite outgrowth and matrix metalloproteinase secretion.
"AG73 has been tested both in vitro and in vivo in different cells and systems (Engbring et al., 2002, in press; Hoffman et al., 2001, 1998). It promotes attachment of numerous cell types (Nomizu et al., 1995, 1998), induces salivary acinar cell differentiation (Hoffman et al., 1998), inhibits branching morphogenesis of embryonic salivary glands (Kadoya et al., 2003, 1998; Kadoya and Yamashina, 2005), stimulates neurite outgrowth (Richard et al., 1996), stimulates matrix metalloproteinase secretion by PC12 cells (Weeks et al., 1998). AG73 has a striking relevance in tumor biology (Engbring et al., 2002, in press; Kim et al., 1998; Mochizuki et al., 2007; Song et al., 1997; Suzuki et al., 2003b, 2005). "
[Show abstract][Hide abstract] ABSTRACT: We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.
"Similar abnormal epithelial cell basal morphology has been seen in corneal and salivary gland epithelia after digesting their basement membranes with proteolytic enzymes ( Sugrue and Hay , 1981 ; Kadoya and Yamashina , 1991 ) . Interestingly , AG - 73 stimu - lates the secretion of 72 kDa matrix metalloprotease in PC - 12 pheochromocytoma cells ( Weeks et al , 1998 ) and 92 kDa matrix metalloprotease in B16 - F10 melanoma cells ( Kim et al , 1998 ) . Matrix metalloproteases play an important part in basement membrane remodeling in many biologic processes , including organogenesis , implantation , and tumor invasion ( Vu and Werb , 2000 ) . "
[Show abstract][Hide abstract] ABSTRACT: We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.
"Metalloproteinase induction was blocked by an antibody directed against the 67-kDa laminin receptor, implicating the YIGSR sequence in proteinase regulation. Peptides containing the IKVAV sequence were also shown to alter type IV collagenase production in a variety of cell types including melanoma cells, macrophages, and neuronal PC12 cells (Corcoran et al. 1995; Royce et al., 1992; Weeks et al, 1998). Treatment of macrophages with IKVAV-containing peptides resulted in activation of the mitogenactivated protein kinase (MAPK) pathway via protein kinase C, suggesting a mechanism for altered proteinase expression (Khan and Falcone, 2000). "
[Show abstract][Hide abstract] ABSTRACT: The laminin family contains a number of complex, multi-domain proteins that participate in a large variety of biologic processes. Limited proteolysis has been utilized extensively as a tool with which to determine laminin structure/function relationships. In addition, proteolytic modification of laminins may occur as a component of heterotrimer assembly and secretion, or may follow incorporation of mature laminin into the extracellular matrix. Conversely, laminin binding to cellular receptors may also influence proteinase expression. This review will highlight specific examples to demonstrate the functional interplay between laminins and proteinases in the regulation of laminin structure and function as well as in the subsequent control of proteinase expression.
Microscopy Research and Technique 11/2000; 51(3):238-46. DOI:10.1002/1097-0029(20001101)51:3<238::AID-JEMT4>3.0.CO;2-3 · 1.15 Impact Factor
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