RESUMO
Purpose: Limbal epithelial stem cells (LSCs), located in the basal layer of the corneal epithelium in the corneal limbus, are vital for maintaining the corneal epithelium. LSCs have a high capacity of self-renewal with increased potential for error-free proliferation and poor differentiation. To date, limited research has focused on unveiling the composition of the limbal stem cell niche, and, more important, on the role the specific stem cell niche may have in LSC differentiation and function. Our work investigates the composition of the extracellular matrix in the LSC niche and how it regulates LSC differentiation and function. Methods: Hyaluronan (HA) is naturally synthesized by hyaluronan synthases (HASs), and vertebrates have the following three types: HAS1, HAS2, and HAS3. Wild-type and HAS and TSG-6 knockout mice-HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, TSG-6-/--were used to determine the importance of the HA niche in LSC differentiation and specification. Results: Our data demonstrate that the LSC niche is composed of a HA rich extracellular matrix. HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, and TSG-6-/- mice have delayed wound healing and increased inflammation after injury. Interestingly, upon insult the HAS knock-out mice up-regulate HA throughout the cornea through a compensatory mechanism, and in turn this alters LSC and epithelial cell specification. Conclusions: The LSC niche is composed of a specialized HA matrix that differs from that present in the rest of the corneal epithelium, and the disruption of this specific HA matrix within the LSC niche leads to compromised corneal epithelial regeneration. Finally, our findings suggest that HA has a major role in maintaining the LSC phenotype.
Assuntos
Diferenciação Celular/fisiologia , Microambiente Celular/fisiologia , Epitélio Corneano/metabolismo , Ácido Hialurônico/metabolismo , Limbo da Córnea/citologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Animais , Queimaduras Químicas/metabolismo , Modelos Animais de Doenças , Queimaduras Oculares/induzido quimicamente , Glucuronosiltransferase/metabolismo , Hialuronan Sintases , Ácido Hialurônico/genética , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Hidróxido de Sódio , Cicatrização/fisiologiaRESUMO
The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/biossíntese , Sulfato de Queratano/biossíntese , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteoglicanas de Sulfatos de Condroitina/deficiência , Humanos , Integrina beta1/metabolismo , Sulfato de Queratano/deficiência , Lumicana , Masculino , Camundongos , Camundongos Knockout , Neoplasias da Próstata/patologia , Regulação para CimaRESUMO
During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.
Assuntos
Colágeno/biossíntese , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação para Cima , Idoso , Linhagem Celular Tumoral , Técnicas de Cocultura , Colágeno/genética , Neoplasias Colorretais/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteoglicanas/metabolismo , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Growth and survival of tumors at a site of metastasis involve interactions with stromal cells in the surrounding environment. Stromal cells aid tumor cell growth by producing cytokines as well as by modifying the environment surrounding the tumor through modulation of the extracellular matrix (ECM). Small leucine-rich proteoglycans (SLRPs) are biologically active components of the ECM which can be altered in the stroma surrounding tumors. The influence tumor cells have on stromal cells has been well elucidated. However, little is understood about the effect metastatic cancer cells have on the cell biology and behavior of the local stromal cells. Our data reveal a significant down-regulation in the expression of ECM components such as collagens I, II, III, and IV, and the SLRPs, decorin, biglycan, lumican, and fibromodulin in stromal cells when grown in the presence of two metastatic prostate cancer cell lines PC3 and DU145. Interestingly, TGF-ß down-regulation was observed in stromal cells, as well as actin depolymerization and increased vimentin and α5ß1 integrin expression. MT1-MMP expression was upregulated and localized in stromal cell protrusions which extended into the ECM. Moreover, enhanced stromal cell migration was observed after cross-talk with metastatic prostate tumor cells. Xenografting metastatic prostate cancer cells together with "activated" stromal cells led to increased tumorigenicity of the prostate cancer cells. Our findings suggest that metastatic prostate cancer cells create a metastatic niche by altering the phenotype of local stromal cells, leading to changes in the ECM.
Assuntos
Diferenciação Celular/genética , Regulação para Baixo/genética , Matriz Extracelular/genética , Fibroblastos/patologia , Neoplasias da Próstata/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/genética , Regulação para Baixo/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Transporte Proteico/efeitos dos fármacos , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this study, we analyzed the influence of proteoglycans on the interaction between human high molecular weight kininogen (HK) and the cell surface. We found that D5- related peptide inhibits HK-biotin cellular uptake. Confocal microscopy showed that HK colocalizes with heparan sulfate proteoglycan (HSPG) at the cell surface. When biotin-HK is incubated with rabbit aorta endothelial cells (RAECs) and CHO-K1 cells, it is internalized into acidic intracellular vesicles, whereas when incubated with CHO-745 cells, which express reduced levels of glycosaminoglycans, HK is not internalized. To further verify the hypothesis that HSPG-dependent mechanisms are involved in HK uptake and proteolytic processing in lysosomes, we tested chloroquine, which blocks Alexa 488- HK colocalization with Lyso Tracker in acidic endosomal vesicles. The process of HK internalization was blocked by low temperatures, methyl-beta-cyclodextrin, FCCP and 2-deoxy-D-glucose, implying that HK uptake into acidic vesicles is energy-dependent and most likely involves binding to HSPG structures localized in cholesterol-rich domains present in the plasma membrane. Kinin generation at the cell surface was much higher in tumorigenic cells (CHO-K1) when compared to endothelial cells (RAECs). The present data indicate that the process of HK endocytosis involving HSPG is a novel additional mechanism which may control kinin generation at the cell surface.
Assuntos
Células Endoteliais/metabolismo , Proteoglicanas de Heparan Sulfato/farmacologia , Cininogênio de Alto Peso Molecular/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Endocitose , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Proteoglicanas , CoelhosRESUMO
Injury to the CNS of vertebrates leads to the formation of a glial scar and production of inhibitory molecules, including chondroitin sulphate proteoglycans. Various studies suggest that the sugar component of the proteoglycan is responsible for the inhibitory role of these compounds in axonal regeneration. By degrading chondroitin sulphate chains with specific enzymes, denominated chondroitinases, the inhibitory capacity of these proteoglycans is decreased. Chondroitinase administration involves frequent injections of the enzyme at the lesion site which constitutes a rather invasive method. We have produced a vector containing the gene for Flavobacterium heparinum chondroitinase AC for expression in adult bone marrow-derived cells which were then transplanted into an injury site in the CNS. The expression and secretion of active chondroitinase AC was observed in vitro using transfected Chinese hamster ovarian and gliosarcoma cells and in vivo by immunohistochemistry analysis which showed degraded chondroitin sulphate coinciding with the location of transfected bone marrow-derived cells. Immunolabelling of the axonal growth-associated protein GAP-43 was observed in vivo and coincided with the location of degraded chondroitin sulphate. We propose that bone marrow-derived mononuclear cells, transfected with our construct and transplanted into CNS, could be a potential tool for studying an alternative chondroitinase AC delivery method.