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17 results on '"Rebustini, Ivan T."'

13. ECM and FGF-dependent assay of embryonic submandibular gland epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during development

Author

Rebustini, Ivan T. and Hoffman, Matthew P.

Subjects
Fibroblast Growth Factors, Mice, Gene Expression Regulation, Submandibular Gland, Cell Adhesion, Animals, Article, Culture Media, Extracellular Matrix
Abstract

Epithelial-mesenchymal interactions during organogenesis are regulated by dynamic and reciprocal interactions between growth factors and extracellular matrix (ECM) components. Mouse embryonic submandibular gland (SMG) epithelium, isolated from its endogenous mesenchyme, undergoes branching morphogenesis when cultured ex vivo in a basement membrane extract in serum-free medium with growth factor stimulation. The resulting three-dimensional epithelial morphogenesis in the defined culture system makes this a useful model to analyze cell-cell and cell-matrix interactions, growth factor-mediated signaling and gene expression, proliferation, apoptosis, migration, lumen formation, and epithelial morphogenesis in a primary organ culture system. SMG epithelial culture is robust, reproducible, uses small amounts of reagents, and changes in gene expression are measured by real-time PCR using a limited amount of embryonic tissue. In this chapter, we describe a detailed protocol for isolating primary embryonic SMG epithelium and setting up an ECM and growth factor-dependent, serum-free assay of epithelial morphogenesis, with subsequent analysis of gene expression by real-time PCR.

Published
2009

14. MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

Author

Rebustini, Ivan T, Myers, Christopher, Lassiter, Keyonica S, Surmak, Andrew, Szabova, Ludmila, Holmbeck, Kenn, Pedchenko, Vadim, Hudson, Billy G, Hoffman, Matthew P, Rebustini, Ivan T, Myers, Christopher, Lassiter, Keyonica S, Surmak, Andrew, Szabova, Ludmila, Holmbeck, Kenn, Pedchenko, Vadim, Hudson, Billy G, and Hoffman, Matthew P

Abstract

Proteolysis is essential during branching morphogenesis, but the roles of MT-MMPs and their proteolytic products are not clearly understood. Here, we discover that decreasing MT-MMP activity during submandibular gland branching morphogenesis decreases proliferation and increases collagen IV and MT-MMP expression. Specifically, reducing epithelial MT2-MMP profoundly decreases proliferation and morphogenesis, increases Col4a2 and intracellular accumulation of collagen IV, and decreases the proteolytic release of collagen IV NC1 domains. Importantly, we demonstrate the presence of collagen IV NC1 domains in developing tissue. Furthermore, recombinant collagen IV NC1 domains rescue branching morphogenesis after MT2-siRNA treatment, increasing MT-MMP and proproliferative gene expression via beta1 integrin and PI3K-AKT signaling. Additionally, HBEGF also rescues MT2-siRNA treatment, increasing NC1 domain release, proliferation, and MT2-MMP and Hbegf expression. Our studies provide mechanistic insight into how MT2-MMP-dependent release of bioactive NC1 domains from collagen IV is critical for integrating collagen IV synthesis and proteolysis with epithelial proliferation during branching morphogenesis.

Published
2009

17. ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during submandibular gland development.

Author

Rebustini IT and Hoffman MP

Subjects
Animals, Cell Adhesion, Culture Media, Mice, Extracellular Matrix metabolism, Fibroblast Growth Factors physiology, Gene Expression Regulation physiology, Submandibular Gland embryology
Abstract

Epithelial-mesenchymal interactions during organogenesis are regulated by dynamic and reciprocal interactions between growth factors and extracellular matrix (ECM) components. Mouse embryonic submandibular gland (SMG) epithelium, isolated from its endogenous mesenchyme, undergoes branching morphogenesis when cultured ex vivo in a basement membrane extract in serum-free medium with growth factor stimulation. The resulting three-dimensional epithelial morphogenesis in the defined culture system makes this a useful model to analyze cell-cell and cell-matrix interactions, growth factor-mediated signaling and gene expression, proliferation, apoptosis, migration, lumen formation, and epithelial morphogenesis in a primary organ culture system. SMG epithelial culture is robust, reproducible, uses small amounts of reagents, and changes in gene expression are measured by real-time PCR using a limited amount of embryonic tissue. In this chapter, we describe a detailed protocol for isolating primary embryonic SMG epithelium and setting up an ECM and growth factor-dependent, serum-free assay of epithelial morphogenesis, with subsequent analysis of gene expression by real-time PCR.

Published
2009
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