FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2015719172> ?p ?o ?g. }

• W2015719172 endingPage "493" @default.
• W2015719172 startingPage "482" @default.
• W2015719172 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 β1 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. 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 β1 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. Membrane-type matrix metalloproteinases (MT-MMPs) are pericellular collagenolytic enzymes responsible for extracellular matrix degradation in many biological processes (Holmbeck et al., 1999Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. et al.MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover.Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar, Hotary et al., 2000Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3.J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (501) Google Scholar, Page-McCaw et al., 2007Page-McCaw A. Ewald A.J. Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling.Nat. Rev. Mol. Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (1993) Google Scholar, Sternlicht and Werb, 2001Sternlicht M.D. Werb Z. How matrix metalloproteinases regulate cell behavior.Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3078) Google Scholar). During branching morphogenesis, cells migrate into the stroma as an epithelial sheet, and both epithelia and stroma produce basement membrane (BM) components as well as proteases that degrade the BM (Bernfield et al., 1984Bernfield M. Banerjee S.D. Koda J.E. Rapraeger A.C. Remodelling of the basement membrane: morphogenesis and maturation.Ciba Found. Symp. 1984; 108: 179-196PubMed Google Scholar, Hogan, 1999Hogan B.L. Morphogenesis.Cell. 1999; 96: 225-233Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, Lu and Werb, 2008Lu P. Werb Z. Patterning mechanisms of branched organs.Science. 2008; 322: 1506-1509Crossref PubMed Scopus (131) Google Scholar, Metzger and Krasnow, 1999Metzger R.J. Krasnow M.A. Genetic control of branching morphogenesis.Science. 1999; 284: 1635-1639Crossref PubMed Scopus (394) Google Scholar). In contrast, during cancer cell invasion, invadopodia perforate the intact BM, and cellular transmigration occurs (Artym et al., 2006Artym V.V. Zhang Y. Seillier-Moiseiwitsch F. Yamada K.M. Mueller S.C. Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function.Cancer Res. 2006; 66: 3034-3043Crossref PubMed Scopus (430) Google Scholar, Hotary et al., 2006Hotary K. Li X.Y. Allen E. Stevens S.L. Weiss S.J. A cancer cell metalloprotease triad regulates the basement membrane transmigration program.Genes Dev. 2006; 20: 2673-2686Crossref PubMed Scopus (282) Google Scholar). However, both branching morphogenesis and cancer cell invasion require MT-MMPs to degrade BM components, generating cleavage products with potential signaling functions and locally releasing growth factors stored in the BM (Ortega and Werb, 2002Ortega N. Werb Z. New functional roles for non-collagenous domains of basement membrane collagens.J. Cell Sci. 2002; 115: 4201-4214Crossref PubMed Scopus (173) Google Scholar). Most single MMP knockouts have subtle developmental phenotypes, which have been explained because of enzymatic redundancy, compensation, and adaptive development (Page-McCaw et al., 2007Page-McCaw A. Ewald A.J. Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling.Nat. Rev. Mol. Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (1993) Google Scholar). However, mice lacking MT1-MMP (Mmp14−/−) have severely impaired collagen metabolism and bone morphogenesis (Holmbeck et al., 1999Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. et al.MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover.Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar, Zhou et al., 2000Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I.Proc. Natl. Acad. Sci. USA. 2000; 97: 4052-4057Crossref PubMed Scopus (659) Google Scholar), lung defects (Atkinson et al., 2005Atkinson J.J. Holmbeck K. Yamada S. Birkedal-Hansen H. Parks W.C. Senior R.M. Membrane-type 1 matrix metalloproteinase is required for normal alveolar development.Dev. Dyn. 2005; 232: 1079-1090Crossref PubMed Scopus (72) Google Scholar), and decreased embryonic submandibular gland (SMG) branching morphogenesis (Oblander et al., 2005Oblander S.A. Zhou Z. Galvez B.G. Starcher B. Shannon J.M. Durbeej M. Arroyo A.G. Tryggvason K. Apte S.S. Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation.Dev. Biol. 2005; 277: 255-269Crossref PubMed Scopus (103) Google Scholar). MT1/MT3-MMP double null mice die at birth with skeletal and craniofacial defects (Shi et al., 2008Shi J. Son M.Y. Yamada S. Szabova L. Kahan S. Chrysovergis K. Wolf L. Surmak A. Holmbeck K. Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis.Dev. Biol. 2008; 313: 196-209Crossref PubMed Scopus (79) Google Scholar). The MMP2/MT1-MMP double null mice also die after birth with respiratory failure and blood vessel and muscle defects (Oh et al., 2004Oh J. Takahashi R. Adachi E. Kondo S. Kuratomi S. Noma A. Alexander D.B. Motoda H. Okada A. Seiki M. et al.Mutations in two matrix metalloproteinase genes, MMP-2 and MT1-MMP, are synthetic lethal in mice.Oncogene. 2004; 23: 5041-5048Crossref PubMed Scopus (104) Google Scholar). However, transcriptional compensation among different MMPs has not been reported in any of these knockout mice. MT-MMPs (referred to as MT1, MT2, and MT3) have overlapping functions and target common substrates, but the differential localization within specific tissues may specify their functions (Page-McCaw et al., 2007Page-McCaw A. Ewald A.J. Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling.Nat. Rev. Mol. Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (1993) Google Scholar, Szabova et al., 2005Szabova L. Yamada S.S. Birkedal-Hansen H. Holmbeck K. Expression pattern of four membrane-type matrix metalloproteinases in the normal and diseased mouse mammary gland.J. Cell. Physiol. 2005; 205: 123-132Crossref PubMed Scopus (38) Google Scholar). MT-MMPs regulate invasion of 3D collagen matrices by MDCK cells, and cancer cell lines overexpressing MT1, MT2, or MT3, but not MMP2 or MMP9, degrade and transmigrate collagen IV-containing ex vivo peritoneal BMs (Hotary et al., 2000Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3.J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (501) Google Scholar, Hotary et al., 2006Hotary K. Li X.Y. Allen E. Stevens S.L. Weiss S.J. A cancer cell metalloprotease triad regulates the basement membrane transmigration program.Genes Dev. 2006; 20: 2673-2686Crossref PubMed Scopus (282) Google Scholar). However, multiple secreted and membrane-type MMPs can degrade collagen IV in other biological contexts (Page-McCaw et al., 2007Page-McCaw A. Ewald A.J. Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling.Nat. Rev. Mol. Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (1993) Google Scholar). MT2 is also expressed in mammary epithelium (Szabova et al., 2005Szabova L. Yamada S.S. Birkedal-Hansen H. Holmbeck K. Expression pattern of four membrane-type matrix metalloproteinases in the normal and diseased mouse mammary gland.J. Cell. Physiol. 2005; 205: 123-132Crossref PubMed Scopus (38) Google Scholar); however, its role during branching morphogenesis had not been investigated. Collagen IV is essential for mouse development, providing structural integrity to the BM, but is not required for deposition or assembly of the BM (Poschl et al., 2004Poschl E. Schlotzer-Schrehardt U. Brachvogel B. Saito K. Ninomiya Y. Mayer U. Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development.Development. 2004; 131: 1619-1628Crossref PubMed Scopus (518) Google Scholar). It is ubiquitously expressed in branching organs (Kuhn, 1995Kuhn K. Basement membrane (type IV) collagen.Matrix Biol. 1995; 14: 439-445Crossref PubMed Scopus (227) Google Scholar, Poschl et al., 2004Poschl E. Schlotzer-Schrehardt U. Brachvogel B. Saito K. Ninomiya Y. Mayer U. Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development.Development. 2004; 131: 1619-1628Crossref PubMed Scopus (518) Google Scholar) as a triple helix containing a 2:1 ratio of col4α1 and col4α2 chains (Borza et al., 2001Borza D.B. Bondar O. Ninomiya Y. Sado Y. Naito I. Todd P. Hudson B.G. The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5, and alpha 6 chains in smooth muscle basement membranes.J. Biol. Chem. 2001; 276: 28532-28540Crossref PubMed Scopus (113) Google Scholar, Khoshnoodi et al., 2008Khoshnoodi J. Pedchenko V. Hudson B.G. Mammalian collagen IV.Microsc. Res. Tech. 2008; 71: 357-370Crossref PubMed Scopus (390) Google Scholar, Vanacore et al., 2004Vanacore R.M. Shanmugasundararaj S. Friedman D.B. Bondar O. Hudson B.G. Sundaramoorthy M. The alpha1.alpha2 network of collagen IV. Reinforced stabilization of the noncollagenous domain-1 by noncovalent forces and the absence of Met-Lys cross-links.J. Biol. Chem. 2004; 279: 44723-44730Crossref PubMed Scopus (39) Google Scholar). MMP-mediated proteolysis of the native triple helix of collagen IV releases NC1 domains, and although NC1 domains have not been previously isolated from developing tissue, the recombinant NC1 domains have distinct signaling properties (Mundel and Kalluri, 2007Mundel T.M. Kalluri R. Type IV collagen-derived angiogenesis inhibitors.Microvasc. Res. 2007; 74: 85-89Crossref PubMed Scopus (139) Google Scholar, Sternlicht and Werb, 2001Sternlicht M.D. Werb Z. How matrix metalloproteinases regulate cell behavior.Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3078) Google Scholar). The capacity of exogenous NC1 domains to block tissue development in vivo was first described in hydra (Zhang et al., 1994Zhang X. Hudson B.G. Sarras Jr., M.P. Hydra cell aggregate development is blocked by selective fragments of fibronectin and type IV collagen.Dev. Biol. 1994; 164: 10-23Crossref PubMed Scopus (51) Google Scholar). Recombinant α1(IV)NC1 and α2(IV)NC1 are antiangiogenic (Petitclerc et al., 2000Petitclerc E. Boutaud A. Prestayko A. Xu J. Sado Y. Ninomiya Y. Sarras Jr., M.P. Hudson B.G. Brooks P.C. New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo.J. Biol. Chem. 2000; 275: 8051-8061Crossref PubMed Scopus (266) Google Scholar, Roth et al., 2005Roth J.M. Akalu A. Zelmanovich A. Policarpio D. Ng B. MacDonald S. Formenti S. Liebes L. Brooks P.C. Recombinant alpha2(IV)NC1 domain inhibits tumor cell-extracellular matrix interactions, induces cellular senescence, and inhibits tumor growth in vivo.Am. J. Pathol. 2005; 166: 901-911Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and bind β1-containing integrins (Khoshnoodi et al., 2008Khoshnoodi J. Pedchenko V. Hudson B.G. Mammalian collagen IV.Microsc. Res. Tech. 2008; 71: 357-370Crossref PubMed Scopus (390) Google Scholar), but whether they regulate MT-MMP expression and epithelial proliferation during branching morphogenesis is unknown. SMG branching morphogenesis requires epithelial proliferation, end bud expansion, and cleft formation and is dependent on local accumulation of extracellular matrix (Patel et al., 2006Patel V.N. Rebustini I.T. Hoffman M.P. Salivary gland branching morphogenesis.Differentiation. 2006; 74: 349-364Crossref PubMed Scopus (211) Google Scholar). Cleft formation involves accumulation and turnover of interstitial collagen fibers (Fukuda et al., 1988Fukuda Y. Masuda Y. Kishi J. Hashimoto Y. Hayakawa T. Nogawa H. Nakanishi Y. The role of interstitial collagens in cleft formation of mouse embryonic submandibular gland during initial branching.Development. 1988; 103: 259-267PubMed Google Scholar, Hayakawa et al., 1992Hayakawa T. Kishi J. Nakanishi Y. Salivary gland morphogenesis: possible involvement of collagenase.Matrix Suppl. 1992; 1: 344-351PubMed Google Scholar), and fibrillar collagens I and III are substrates for MT1 and MT3 (d'Ortho et al., 1997d'Ortho M.P. Will H. Atkinson S. Butler G. Messent A. Gavrilovic J. Smith B. Timpl R. Zardi L. Murphy G. Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases.Eur. J. Biochem. 1997; 250: 751-757Crossref PubMed Scopus (374) Google Scholar). More recently, important roles for the accumulation and localization of other ECM components in epithelial clefts, such as laminins, fibronectin, and perlecan, have been reported (Patel et al., 2007Patel V.N. Knox S.M. Likar K.M. Lathrop C.A. Hossain R. Eftekhari S. Whitelock J.M. Elkin M. Vlodavsky I. Hoffman M.P. Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis.Development. 2007; 134: 4177-4186Crossref PubMed Scopus (121) Google Scholar, Rebustini et al., 2007Rebustini I.T. Patel V.N. Stewart J.S. Layvey A. Georges-Labouesse E. Miner J.H. Hoffman M.P. Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling.Dev. Biol. 2007; 308: 15-29Crossref PubMed Scopus (85) Google Scholar, Sakai et al., 2003Sakai T. Larsen M. Yamada K.M. Fibronectin requirement in branching morphogenesis.Nature. 2003; 423: 876-881Crossref PubMed Scopus (388) Google Scholar). Nonetheless, the role of collagen IV synthesis and proteolysis and the MT-MMPs involved during branching morphogenesis are still unknown. Here, we investigate MT2-dependent proteolysis of collagen IV during branching morphogenesis. Knockdown of MT2 in explant culture reduces cell proliferation and morphogenesis and disrupts collagen IV metabolism, resulting in intracellular accumulation of collagen IV and reduced production of NC1 domains. We propose that the proteolytic release of NC1 domains plays a critical role in branching morphogenesis by regulating further protease expression and epithelial proliferation. A broad MMP inhibitor (GM6001) inhibits E13 SMG branching, resulting in enlarged end buds, decreased epithelial cell proliferation, a striking accumulation of collagen IV, and reduced end bud number (Figure 1A, insert graph in Figure 1B). The collagen IV accumulation is around both epithelium and mesenchyme, but the mesenchymal accumulation is most apparent in the single projection of a confocal stack of images (Figure 1A). GM6001 also increased Col4a2 8-fold and Col4a1 2-fold, whereas Lama5, Hspg2, MT1, MT2, and MT3 did not change (Figure 1B). Gelatin zymography of the conditioned medium showed that GM6001 inhibited secreted MMP2 activation in a time-dependent manner (see Figure S1A available online). We also compared the effects of exogenous TIMP1 with TIMP2, which inhibits both secreted and MT1-, 2-, and 3-MMPs. TIMP2 but not TIMP1 significantly decreased E13 SMG branching (Figure 1C). In addition, the expression of MT2, MT3, and Col4a2 increased after TIMP2 inhibition (Figure 1C). Taken together, our results highlight the central role of membrane-type, rather than secreted MMPs, during SMG morphogenesis. This suggests that a proteolytic product of MT-MMPs may regulate epithelial proliferation and result in transcriptional feedback to MT-MMP and collagen IV expression. The SMGs of MT1−/− mice undergo reduced branching morphogenesis in culture, but branching of the lungs was not altered (Oblander et al., 2005Oblander S.A. Zhou Z. Galvez B.G. Starcher B. Shannon J.M. Durbeej M. Arroyo A.G. Tryggvason K. Apte S.S. Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation.Dev. Biol. 2005; 277: 255-269Crossref PubMed Scopus (103) Google Scholar), suggesting that MT-MMPs may have specific functions in the SMG. Transcriptional compensation by other MT-MMPs was not reported; therefore, tissue-specific differences in MT-MMP function may also occur. When we cultured MT1+/+, MT1+/−, and MT1−/− SMGs, we confirmed that there was decreased branching in MT1−/− SMGs and also discovered there was reduced branching in MT1+/− compared to MT1+/+ SMGs (Figure 1D, pictures and insert graph). Function-blocking antibodies to MT1-MMP also reduced SMG branching (Figure S1B). We confirmed the reduction and the absence of MT1 expression in the MT1+/− and MT1−/− SMGs, respectively. However, we unexpectedly detected a specific increase of MT2 in both MT1+/− (6.4-fold) and MT1−/− (8.2-fold), compared to MT1+/+ SMGs. (Figure 1D). The data suggest that a transcriptional compensation of MT2 expression occurs with reduced MT1 expression in vivo, and since exogenous TIMP2 also increased MT2 and MT3 expression (Figure 1C), we investigated the spatiotemporal expression of the MT-MMPs during SMG development. We analyzed the expression of MT-MMPs and collagen IV throughout SMG development. MT1, MT2, MT3, Mmps2, -9, and -11, Col4a1, and Col4a2 were upregulated at E13, and Mmps19 and -23 were also present (Figure 2A and Figure S2). We separated E13 epithelium from the mesenchyme and analyzed MMP expression by qPCR. MT2 was more abundant in the epithelium, with MT1 and MT3 more abundant in the mesenchyme (Figure 2B). Whole-mount immunofluorescent analysis confirmed the predominant epithelial localization of MT2, whereas MT3 was present in both epithelium and mesenchyme, and MT1 accumulated in the mesenchyme around cleft regions of the epithelium (Figure 2C). The specificity of the MT-MMP antibodies was confirmed using MT1−/− and MT3−/− SMGs or by using the immunizing peptide to compete the MT2 antibody (Figures S3A–S3C). The compensatory increases in MT-MMP expression (Figure 1, Figure 2) were further investigated using siRNAs to downregulate MT1, MT2, and MT3 in SMG explant cultures. MT1-siRNA decreased branching (Figure 3A), with a phenotype similar to the MT1+/− and MT1−/− SMGs (Figure 1D). Intriguingly, MT2-siRNA resulted in the most severe disruption of branching: the end buds enlarged but did not completely form clefts, and secondary duct formation was delayed (Figure 3A). In contrast, MT3-siRNA did not affect branching, and the SMGs appeared similar to NS-siRNA treatment. All siRNA treatments decreased the expression of their corresponding targeted mRNA and protein by ∼50%, as measured by qPCR (Figure 3B) and whole-mount immunostaining, respectively (Figure S3D). MT2-siRNA treatment resulted in a 4.5-fold increase in Col4a2 and a 2.2-fold increase in Lama5; MT1-siRNA and MT3-siRNA treatments resulted in a 1.8-fold and a 6.2-fold increase in MT2 expression, respectively. There was less transcriptional increase of MT2 with MT1-siRNA (Figure 3B) compared to MT1+/− SMGs (Figure 1D), most likely because the genetic reduction in MT1+/− SMGs occurs earlier in development, whereas the knockdown in explant culture occurs with existing proteolytic products present. Additionally, the decrease in branching morphogenesis with MT2-siRNA treatment was rescued by exogenous recombinant MT2- and MT3-MMP catalytic domains (recMT2 and recMT3) but not recMT1 (Figure S4A). The recMTs are functional in vitro, and we treated both SMG collagen IV and bovine lens basement membrane with the recMTs and show they release NC1 domains (Figures S4D and S4E). We also decreased MT-MMP expression directly in isolated SMG epithelia cultured in a 3D laminin-111 ECM with FGF10 (Steinberg et al., 2005Steinberg Z. Myers C. Heim V.M. Lathrop C.A. Rebustini I.T. Stewart J.S. Larsen M. Hoffman M.P. FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.Development. 2005; 132: 1223-1234Crossref PubMed Scopus (180) Google Scholar). MT2-siRNA had the greatest effect on epithelial morphogenesis; MT1- (which is not highly expressed in the epithelium) and MT3-siRNA did not decrease epithelial morphogenesis and were similar to the NS-siRNA control (Figure 3C). Epithelial morphogenesis was expressed as a morphogenic index (number of end buds × duct length, in AU ×103), which was significantly decreased with MT2-siRNA (Figure 3C, graph). Analysis of gene expression in the MT2-siRNA-treated epithelia showed a significant decrease of MT2 expression and a 3-fold increase of Col4a2 expression (Figure 3D). Epithelial morphogenesis with MT2-siRNA was also rescued by recMT2 (Figure S4B). The ability of recMT2 to activate pro-MMP2 was confirmed by gelatin zymography of the culture media after siRNA and recMT2 treatment (Figure S4C). Taken together, these experiments indicate that there is a coordinated transcriptional regulation of MT-MMPs: reducing MT1 or MT3 expression upregulates MT2, while reducing MT2 expression upregulates MT3 expression. In addition, MT2-siRNA has the greatest effect on SMG epithelial morphogenesis and specifically increases Col4a2 expression. We measured cell proliferation by Ki67 staining and immunolocalized collagen IV after MT-siRNA treatment. MT2-siRNA significantly decreased epithelial cell proliferation, particularly in the end buds (Figure 4A, left panels), whereas NS-, MT1-, and MT3-siRNAs had minimal effects on proliferation (Figure 4B). We observed increased intracellular collagen IV, which was most apparent in the epithelial cells in MT2-siRNA-treated SMGs (Figure 4A, middle panels) as compared to MT1- and MT3-siRNA treatments. The intracellular location was evident when stacks of confocal sections stained with an epithelial cell surface marker, syndecan 4, were analyzed (Figure S5A). Staining with lamp1, a lysosomal marker, showed there was not increased localization of the intracellular collagen IV in lysosomes (Figure S5B). Quantification of collagen IV staining shows that while MT2-siRNA has the greatest effect, all MT-siRNAs increased collagen IV compared to NS-siRNA treatment (Figure 4B). We immunoprecipitated (IP) collagen IV from both SMG lysates and the culture media (data not shown) using a Col4α2 monoclonal antibody and detected Col4α1, Col4α2, and the NC1 domains by Western blot with a polyclonal antibody to collagen IV. Both purified collagen IV and recombinant NC1 domains were used as positive controls. We observed an increase in the intact Col4α1 and Col4α2 with MT1-, MT2-, and MT3-siRNA compared to NS-siRNA, although MT2-siRNA had the greatest increase (upper blot in Figure 4C). Also, the NC1 domains decreased after both MT1- and MT2-siRNA, compared to the NS-siRNA (lower blot in Figure 4C). There was a decrease in NC1 domains released in GM6001- or TIMP2-treated E12 SMGs (Figures S6A and S6B). We were unable to distinguish between α1(IV)NC1 and α2(IV)NC1, as their molecular weights (∼25 kDA) are similar, and the polyclonal antibody recognizes both of them (Figure S6B). Taken together, these findings indicate that decreasing epithelial MT2 expression decreases epithelial cell proliferation, increases intracellular collagen IV accumulation, and decreases proteolytic release of NC1 domains. NC1 domains, released by proteolysis of collagen IV chains, bind to integrin receptors and inhibit angiogenesis (Mundel and Kalluri, 2007Mundel T.M. Kalluri R. Type IV collagen-derived angiogenesis inhibitors.Microvasc. Res. 2007; 74: 85-89Crossref PubMed Scopus (139) Google Scholar, Ortega and Werb, 2002Ortega N. Werb Z. New functional roles for non-collagenous domains of basement membrane collagens.J. Cell Sci. 2002; 115: 4201-4214Crossref PubMed Scopus (173) Google Scholar). We added recombinant α1(IV)NC1 and α2(IV)NC1 domains to SMGs treated with MT2-siRNA. The NC1 domains increased epithelial branching and MT2 expression (Figure 5A), increased cell proliferation, and decreased collagen IV accumulation in the epithelium (Figure 5B). NC1 domains also rescued epithelial morphogenesis after MT1-siRNA treatment in E12 SMG culture (Figures S7A and S7B). Finally, we isolated endogenous NC1 domains by IP directly from E13 SMGs, and these also rescued branching morphogenesis after MT2-siRNA treatment (Figure S8), showing that both the endogenous and recombinant NC1 domains have similar activity. In order to determine how the NC1 domains regulate protease or BM synthesis and epithelial proliferation, we analyzed the expression of MMPs and BM components 4 hr after treatment of E13 SMGs with NC1 domains (Figure 5C). MT1, MT2, MT3, and Col4a2 were all upregulated, whereas Col4a1 did not change. We also measured expression of proproliferative genes involved in SMG morphogenesis such as FGFR (Hoffman et al., 2002Hoffman M.P. Kidder B.L. Steinberg Z.L. Lakhani S. Ho S. Kleinman H.K. Larsen M. Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms.Development. 2002; 129: 5767-5778Crossref PubMed Scopus (155) Google Scholar, Steinberg et al., 2005Steinberg Z. Myers C. Heim V.M. Lathrop C.A. Rebustini I.T. Stewart J.S. Larsen M. Hoffman M.P. FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.Development. 2005; 132: 1223-1234Crossref PubMed Scopus (180) Google Scholar) and EGFR (Kashimata et al., 2000Kashimata M.W. Sakagami H.W. Gresik E.W. Intracellular signalling cascades activated by the EGF receptor and/or by integrins, with potential relevance for branching morphogenesis of the fetal mouse submandibular gland.Eur. J. Morphol. 2000; 38: 269-275Crossref PubMed Google Scholar, Koyama et al., 2008Koyama N. Hayashi T. Ohno K. Siu L. Gresik E.W. Kashimata M. Signaling pathways activated by epidermal growth factor receptor or fibroblast growth factor receptor differentially regulate branching morphogenesis in fetal mouse submandibular glands.Dev. Growth Differ. 2008; 50: 565-576PubMed Google Scholar) signaling components. Fgfr1b, Fgfr2b, Fgf1, and Hbegf increased with 4 hr of NC1 treatment (Figure 5C), whereas Egfr, ErbB2, and ErbB3 increased 24 hr after NC1 treatment (data not shown), downstream of the early transcriptional changes. We then used the induction of MT2 expression by NC1 domains as an assay to investigate the receptors and downstream signaling from NC1 domains. Integrins are receptors for NC1 domains, and their downstream signaling pathways likely regulate the NC1-dependent MT2 transcription. Function-blocking β1 integrin antibodies also inhibit SMG branching morphogenesis (Rebustini et al., 2007Rebustini I.T. Patel V.N. Stewart J.S. Layvey A. Georges-Labouesse E. Miner J.H. Hoffman M.P. Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling.Dev. Biol. 2007; 308: 15-" @default.
• W2015719172 created "2016-06-24" @default.
• W2015719172 creator A5000288286 @default.
• W2015719172 creator A5013320331 @default.
• W2015719172 creator A5020349785 @default.
• W2015719172 creator A5023330084 @default.
• W2015719172 creator A5028874325 @default.
• W2015719172 creator A5031623367 @default.
• W2015719172 creator A5069774314 @default.
• W2015719172 creator A5073653879 @default.
• W2015719172 creator A5090550074 @default.
• W2015719172 date "2009-10-01" @default.
• W2015719172 modified "2023-10-16" @default.
• W2015719172 title "MT2-MMP-Dependent Release of Collagen IV NC1 Domains Regulates Submandibular Gland Branching Morphogenesis" @default.
• W2015719172 cites W1521876081 @default.
• W2015719172 cites W1963818935 @default.
• W2015719172 cites W1966605681 @default.
• W2015719172 cites W1968084778 @default.
• W2015719172 cites W1971441895 @default.
• W2015719172 cites W1972325477 @default.
• W2015719172 cites W1979769719 @default.
• W2015719172 cites W1982351756 @default.
• W2015719172 cites W1985447634 @default.
• W2015719172 cites W1988973424 @default.
• W2015719172 cites W1989847998 @default.
• W2015719172 cites W1991888662 @default.
• W2015719172 cites W2012083187 @default.
• W2015719172 cites W2016484384 @default.
• W2015719172 cites W2026038159 @default.
• W2015719172 cites W2027648896 @default.
• W2015719172 cites W2035991981 @default.
• W2015719172 cites W2044927450 @default.
• W2015719172 cites W2050344009 @default.
• W2015719172 cites W2050778467 @default.
• W2015719172 cites W2051062818 @default.
• W2015719172 cites W2061423797 @default.
• W2015719172 cites W2065039664 @default.
• W2015719172 cites W2071359561 @default.
• W2015719172 cites W2073007695 @default.
• W2015719172 cites W2074744775 @default.
• W2015719172 cites W2076608386 @default.
• W2015719172 cites W2077183324 @default.
• W2015719172 cites W2077955277 @default.
• W2015719172 cites W2086200882 @default.
• W2015719172 cites W2087267111 @default.
• W2015719172 cites W2088019834 @default.
• W2015719172 cites W2089039470 @default.
• W2015719172 cites W2093690358 @default.
• W2015719172 cites W2094842696 @default.
• W2015719172 cites W2096252197 @default.
• W2015719172 cites W2100174776 @default.
• W2015719172 cites W2103212285 @default.
• W2015719172 cites W2107819345 @default.
• W2015719172 cites W2110223227 @default.
• W2015719172 cites W2115810495 @default.
• W2015719172 cites W2118523009 @default.
• W2015719172 cites W2125743967 @default.
• W2015719172 cites W2128049766 @default.
• W2015719172 cites W2128333867 @default.
• W2015719172 cites W2143156113 @default.
• W2015719172 cites W2152485690 @default.
• W2015719172 cites W2158601293 @default.
• W2015719172 cites W2159944716 @default.
• W2015719172 cites W2161354741 @default.
• W2015719172 cites W2165663357 @default.
• W2015719172 cites W2295302139 @default.
• W2015719172 cites W4247817378 @default.
• W2015719172 doi "https://doi.org/10.1016/j.devcel.2009.07.016" @default.
• W2015719172 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2768621" @default.
• W2015719172 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19853562" @default.
• W2015719172 hasPublicationYear "2009" @default.
• W2015719172 type Work @default.
• W2015719172 sameAs 2015719172 @default.
• W2015719172 citedByCount "117" @default.
• W2015719172 countsByYear W20157191722012 @default.
• W2015719172 countsByYear W20157191722013 @default.
• W2015719172 countsByYear W20157191722014 @default.
• W2015719172 countsByYear W20157191722015 @default.
• W2015719172 countsByYear W20157191722016 @default.
• W2015719172 countsByYear W20157191722017 @default.
• W2015719172 countsByYear W20157191722018 @default.
• W2015719172 countsByYear W20157191722019 @default.
• W2015719172 countsByYear W20157191722020 @default.
• W2015719172 countsByYear W20157191722021 @default.
• W2015719172 countsByYear W20157191722022 @default.
• W2015719172 countsByYear W20157191722023 @default.
• W2015719172 crossrefType "journal-article" @default.
• W2015719172 hasAuthorship W2015719172A5000288286 @default.
• W2015719172 hasAuthorship W2015719172A5013320331 @default.
• W2015719172 hasAuthorship W2015719172A5020349785 @default.
• W2015719172 hasAuthorship W2015719172A5023330084 @default.
• W2015719172 hasAuthorship W2015719172A5028874325 @default.
• W2015719172 hasAuthorship W2015719172A5031623367 @default.
• W2015719172 hasAuthorship W2015719172A5069774314 @default.
• W2015719172 hasAuthorship W2015719172A5073653879 @default.
• W2015719172 hasAuthorship W2015719172A5090550074 @default.
• W2015719172 hasBestOaLocation W20157191721 @default.
• W2015719172 hasConcept C104317684 @default.

• next