Detection and purification of instructive extracellular matrix components with monoclonal antibody technologies

Gil, Susana G.; Sigle, Randy O.; Carter, William G.

doi:10.1016/s0091-679x(02)69006-5

Cited by 7 publications

(11 citation statements)

References 54 publications

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“…Tegaderm™ was cut into 1 cm squares, placed in 24-well Petri plates and incubated with 250μL of the monoclonal antibody C2-5 (10μg/mL) at 4°C for 24 h. The C2-5 coated Tegaderm™ (C2-5 biomaterial) was rinsed three times with PBS then blocked with sterile heat-denatured BSA (5 mg/ml in PBS) at 4°C for 24 h. The C2-5 biomaterial was then incubated with 200μL of LM-332 (10 μg/ml) at 4°C for 24 h. The affinity of C2-5 for the amino terminal of the α3 chain of LM-332 ( Figure 1) immobilizes LM-332 on the C2-5 biomaterial by antibody trapping (42) to form a LM-332 biomaterial. C2-5 bound to LM-332 induces keratinocyte adhesion, spreading, and phosphorylation of focal adhesion kinases (42). The LM-332 biomaterial was rinsed three times with PBS then blocked with sterile heat-denatured BSA (5 mg/ml in PBS) at 4°C for 24 h. Presence of C2-5 and C2-5/ LM-332 on Tegaderm™ was confirmed by immunostaining.…”

Section: Lm-332 Biomaterialsmentioning

confidence: 99%

Topical application of laminin-332 to diabetic mouse wounds

Sullivan

Underwood

Sigle

et al. 2007

Journal of Dermatological Science

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Section: Lm-332 Biomaterialsmentioning

confidence: 99%

Topical application of laminin-332 to diabetic mouse wounds

Sullivan

Underwood

Sigle

et al. 2007

Journal of Dermatological Science

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“…Laminin 5-coated surfaces were prepared as previously described (Gil et al, 2002;Xia et al, 1996). Collagen surfaces were prepared by adsorbing human placental type I collagen (Wayner and Carter, 1987) at 10 μg/ml for 2 hours at 24°C.…”

Section: Human Keratinocyte Scrape Wounds In Vitromentioning

confidence: 99%

“…The BSA adhesion assay couples deposition of laminin 5 to subsequent adhesion to the deposits and was performed as previously described (Frank, 2004;Gil et al, 2002). MKs from laminin 5 null mice (α3 -/-MKs) and from a wild-type mouse (α3 +/+ MKs) were grown to confluence, suspended and re-plated onto petri dishes coated with HD-BSA, a non-adhesive surface, or onto laminin 5 surfaces.…”

Section: Bsa Adhesion Assaymentioning

confidence: 99%

“…10). MKs with the wild-type α3 chain of laminin 5 (α3 +/+ MKs) and mutants null for the α3 chain of laminin 5 (α3 -/-MKs) were compared for deposition of laminin 5 and adhesion to the deposits in the BSA adhesion assay (Gil et al, 2002;Sigle et al, 2004). The α3 +/+ and α3 -/-MKs were suspended and re-plated onto either a non-adhesive surface coated with BSA or an adhesive laminin 5 surface (Fig.…”

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confidence: 99%

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Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes

Harper

Alvares

Carter

2005

Journal of Cell Science

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Quiescent epidermis anchors to laminin 5 in the basement membrane via integrin α6β4. Wounding elevates expression of laminin 5, generating leading keratinocytes (LKs) that migrate via β1 integrins. Laminin 5 was evaluated as a regulator of cell signaling, and mRNA and protein expression in LKs. An in vitro wound model was developed based on suspension and re-adhesion of quiescent human keratinocytes (HKs). DNA microarrays identified multiple mRNAs elevated 1.5 hours after suspension and re-adhesion including activation transcription factor 3 (ATF3). In vitro and in vivo, levels of ATF3 protein elevate in nuclei of LKs, but not in nuclei of the following cells, 2 hours after suspension or wounding but decline by 12-18 hours post injury. Significantly, null defects in laminin 5 or integrin β4 that inhibit anchorage chronically elevate ATF3 in vivo. This suggests that adhesion to laminin 5, but not other ligands, suppresses activation. On suspension, ATF3 and other transcripts in the microarrays are elevated by phosphorylated p38 mitogen-activated protein kinase (P-p38), a stress kinase that regulates mRNA and cell motility. Inhibition of P-p38 with SB203580 prevents phosphorylation of ATF2, a transcription factor for ATF3 in LKs. Re-adhesion to laminin 5 via α6β4 dephosphorylates P-p38 and suppresses ATF3 protein relative to cells in suspension. Thus, wounding of quiescent HKs disrupts laminin 5 adhesion to activate p38, generating mRNA transcripts that define LKs. Adhesion to deposits of laminin 5 via α6β4 suppresses P-p38 and activation mRNAs including ATF3. Defects in laminin 5 and α6β4 sustain P-p38 with probable pathological effects on transcription and migration. Journal of Cell Science 3472 of polarized lamellipodium in epithelial cells through activation of Rac1 (Choma et al., 2004). This sequence of adhesion and signaling changes allows HKs to make the transition from quiescence to activation in wounds. Similarly, ligation of α3β1 in A549 adenocarcinoma cells by laminin 10/11 or laminin 5 preferentially activates the Rac-MKK-JNK/p38 stress pathway that mediates epithelial migration (Gu et al., 2001;Ono and Han, 2000;Xia and Karin, 2004). In addition to adhesion, interaction of cells with integrin ligands also regulates mRNA transcription and protein translation through activation of p38 MAPK (Balda and Matter, 2003). Ligation of α6β4 activates the Rac-PAK-MKK-p38 pathway to promote expression of IL-6 in thymic epithelial cells (Mainiero et al., 2003). Thus, cell suspension and re-adhesion onto dermal collagen or laminin 5 via integrins activates JNK and/or p38 by phosphorylation to alter protein expression and/or cell motility (Clark et al., 2003;Ono and Han, 2000). Here, we sought to: (1) identify changes in mRNA transcription and protein translation in quiescent keratinocytes as they transition into wound LKs; (2) identify cell signals necessary for the transcriptional changes in LKs; (3) understand the role of laminin 5 adhesion in regulating the signaling and transcriptional changes. We found that sus...

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“…An antibody against laminin β1 and γ1 chains (rabbit polyclonal antibody R5922) was prepared against denatured β1 and γ1 chains and is able to immunoblot denatured β1 or γ1 chains and immunoprecipitate denatured or nondenatured β1 or γ1 chains in laminins 1 and 6. Mouse Mabs P1H5 (inhibitory anti-α2 integrin), P1B5 (inhibitory anti-α3 integrin), P4C10 (inhibitory anti-β1 integrin), D3-4 (anti-human and mouse laminin α3), C2-5 (antihuman laminin α3), B4-6 (anti-human laminin γ2), C2-9 (inhibitory anti-human laminin α3) and D2-1 (anti-human laminin α3 G4/5 domain) have been described (Gil et al, 1994;Gil et al, 2002;Wayner and Carter, 1987). R5808-f1, a rabbit Mab directed against a peptide sequence (n-EQVYLGLSPSRKSKSLP-c) from the G5 domains of mouse laminin α3 chain was developed in this lab in conjunction with Elizabeth Wayner in the FHCRC hybridoma facility.…”

Section: Antibodiesmentioning

confidence: 99%

Globular domains 4/5 of the laminin α3 chain mediate deposition of precursor laminin 5

Sigle

Gil

Bhattacharya

et al. 2004

Journal of Cell Science

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In epidermal wounds, precursor laminin 5 (α3β3γ2) is deposited in the provisional basement membrane (PBM) before other BM components. Precursor laminin 5 contains G4/5 globular domains at the carboxyl terminus of the α3 chain. Here, the function of G4/5 was evaluated in deposition of laminin 5. Soluble laminin 5, secreted by keratinocytes in culture, is cleaved by an endogenous protease releasing G4/5. Thrombin, a serum protease, cleaves G4/5 indistinguishably from endogenous protease. Soluble human precursor laminin 5, but not cleaved laminin 5, was bound and deposited by mouse keratinocytes null for mouse α3 chain (α3–/– MKs). The deposition rescued adhesion and spreading and survival. In a model for PBM assembly, precursor laminin 5 was deposited along fibronectin fibrils at the junction between co-cultures of keratinocytes and fibroblasts. In both models, the deposition of precursor laminin 5 was inhibited by removal of G4/5 with thrombin. To confirm that G4/5 participates in deposition, the human LAMA3A gene was modified to produce α3 chains either without or with G4/5 that cannot be cleaved. Both precleaved and noncleavable α3 isoforms were expressed in α3–/– MKs, where they deposited sufficiently to rescue adhesion via integrins α3β1 and α6β4. Despite this similarity, noncleavable laminin 5 was at least threefold more efficiently deposited than precleaved isoform. We conclude that the G4/5 domain in the α3 chain facilitates deposition of precursor laminin 5 into the PBM in epidermal wounds.

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Detection and purification of instructive extracellular matrix components with monoclonal antibody technologies

Cited by 7 publications

References 54 publications

Topical application of laminin-332 to diabetic mouse wounds

Topical application of laminin-332 to diabetic mouse wounds

Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes

Globular domains 4/5 of the laminin α3 chain mediate deposition of precursor laminin 5

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