RESUMO
Hyaluronan accumulates in the stroma of several solid tumors and promotes their progression. Both enhanced synthesis and fragmentation of hyaluronan are required as a part of this inflammatory process resembling wound healing. Increased expression of the genes of hyaluronan synthases (HAS1-3) are infrequent in human tumors, while posttranslational modifications that activate the HAS enzymes, and glucose shunted to the UDP-sugar substrates HASs, can have crucial contributions to tumor hyaluronan synthesis. The pericellular hyaluronan influences virtually all cell-cell and cell-matrix interactions, controlling migration, proliferation, apoptosis, epithelial to mesenchymal transition, and stem cell functions. The catabolism by hyaluronidases and free radicals appears to be as important as synthesis for the inflammation that promotes tumor growth, since the receptors mediating the signals create specific responses to hyaluronan fragments. Targeting hyaluronan metabolism shows therapeutic efficiency in animal experiments and early clinical trials.
Assuntos
Hialuronan Sintases/metabolismo , Ácido Hialurônico/biossíntese , Neoplasias/metabolismo , Animais , Comunicação Celular , Movimento Celular , Progressão da Doença , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Transdução de Sinais , Microambiente TumoralRESUMO
Extracellular nucleotides are used as signaling molecules by several cell types. In epidermis, their release is triggered by insults such as ultraviolet radiation, barrier disruption, and tissue wounding, and by specific nerve terminals firing. Increased synthesis of hyaluronan, a ubiquitous extracellular matrix glycosaminoglycan, also occurs in response to stress, leading to the attractive hypothesis that nucleotide signaling and hyaluronan synthesis could also be linked. In HaCaT keratinocytes, ATP caused a rapid and strong but transient activation of hyaluronan synthase 2 (HAS2) expression via protein kinase C-, Ca2+/calmodulin-dependent protein kinase II-, mitogen-activated protein kinase-, and calcium response element-binding protein-dependent pathways by activating the purinergic P2Y2 receptor. Smaller but more persistent up-regulation of HAS3 and CD44, and delayed up-regulation of HAS1 were also observed. Accumulation of peri- and extracellular hyaluronan followed 4-6â h after stimulation, an effect further enhanced by the hyaluronan precursor glucosamine. AMP and adenosine, the degradation products of ATP, markedly inhibited HAS2 expression and, despite concomitant up-regulation of HAS1 and HAS3, inhibited hyaluronan synthesis. Functionally, ATP moderately increased cell migration, whereas AMP and adenosine had no effect. Our data highlight the strong influence of adenosinergic signaling on hyaluronan metabolism in human keratinocytes. Epidermal insults are associated with extracellular ATP release, as well as rapid up-regulation of HAS2/3, CD44, and hyaluronan synthesis, and we show here that the two phenomena are linked. Furthermore, as ATP is rapidly degraded, the opposite effects of its less phosphorylated derivatives facilitate a rapid shut-off of the hyaluronan response, providing a feedback mechanism to prevent excessive reactions when more persistent signals are absent.
Assuntos
Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Epiderme/enzimologia , Hialuronan Sintases/metabolismo , Queratinócitos/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Epiderme/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Hialuronan Sintases/genética , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Receptores Purinérgicos P2Y2/genética , Transdução de SinaisRESUMO
The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 µm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.
Assuntos
Glucuronosiltransferase/metabolismo , Ácido Hialurônico/metabolismo , Queratinócitos/metabolismo , Uridina Trifosfato/metabolismo , Linhagem Celular , Glucuronosiltransferase/genética , Humanos , Hialuronan Sintases , Regulação para CimaRESUMO
UDP-N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.
Assuntos
Aldose-Cetose Isomerases/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Hexosaminas/biossíntese , Hialuronan Sintases/genética , Uridina Difosfato N-Acetilglicosamina/metabolismo , Aldose-Cetose Isomerases/antagonistas & inibidores , Movimento Celular/genética , Frutosefosfatos/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucose/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Humanos , Ácido Hialurônico/biossíntese , Queratinócitos/metabolismo , RNA Interferente Pequeno/genética , Uridina Difosfato N-Acetilglicosamina/genéticaRESUMO
OBJECTIVE: Hyaluronidases (HYAL1 and HYAL2) are key enzymes in the degradation of hyaluronan, and their expression has been altered in various cancer types. We previously showed that hyaluronan accumulation in endometrial carcinomas was correlated with decreased mRNA expression of the HYAL genes. In this study, we analyzed HYAL1 and HYAL2 protein expressions in normal and precancerous endometrial tissues and in endometrial carcinomas. We also investigated whether the protein levels were associated with clinicopathological factors, invasion, and disease recurrence. METHODS: A total of 343 tissue specimens from normal, atrophic, hypertrophic, and neoplastic endometria were analyzed immunohistochemically for HYAL1 and HYAL2 expressions. The results were correlated with clinicopathological factors, the expression of the epithelial-mesenchymal transition marker, E-cadherin, and disease recurrence. RESULTS: Reduced HYAL1 expression was associated with the progression of endometrial carcinomas towards higher grades and also with large tumor sizes, lymph node metastasis, and lymphovascular invasion. Reduced expression of both HYAL1 and HYAL2 was associated with deep myometrial invasion. HYAL2 expression was primarily constant in neoplastic tissues, but its expression was altered in different phases of the endometrial cycle. In addition, a reduction in HYAL1 expression was associated with the depletion of E-cadherin. In a multivariate analysis, reduced HYAL1 expression was an independent prognostic factor for early disease recurrence (HR 5.13, 95% CI: 1.131-23.270, p=0.034). CONCLUSIONS: This study showed that reduced HYAL1 expression was associated with endometrial carcinoma aggressiveness, which further supported the role of hyaluronan degradation in cancer progression.
Assuntos
Neoplasias do Endométrio/enzimologia , Hialuronoglucosaminidase/biossíntese , Recidiva Local de Neoplasia/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologiaRESUMO
Inflammatory pathway plays an important role in tumor cell progression of colorectal cancers. Although colon cancer is considered as one of the leading causes of death worldwide, very few drugs are available for its effective treatment. Many studies have examined the effects of specific COX-2 and 5-LOX inhibitors on human colorectal cancer, but the role of isothiocyanates (ITSCs) as COX-LOX dual inhibitors engaged in hyaluronan-CD44 interaction has not been studied. In the present work, we report series of ITSC analogs incorporating bioisosteric thiosemicarbazone moiety. These inhibitors are effective against panel of human colon cancer cell lines including COX-2 positive HCA-7, HT-29 cells lines, and hyaluronan synthase-2 (Has2) enzyme over-expressing transformed intestinal epithelial Apc10.1Has2 cells. Specifically, our findings indicate that HA-CD44v6-mediated COX-2/5-LOX signaling mediate survivin production, which in turn, supports anti-apoptosis and chemo-resistance leading to colon cancer cell survival. The over-expression of CD44v6shRNA as well as ITSC treatment significantly decreases the survival of colon cancer cells. The present results thus offer an opportunity to evolve potent inhibitors of HA synthesis and CD44v6 pathway and thus underscoring the importance of the ITSC analogs as chemopreventive agents for targeting HA/CD44v6 pathway.
RESUMO
Hyaluronan, a major matrix molecule in epidermis, is often increased by stimuli that enhance keratinocyte proliferation and migration. We found that small amounts of UDP-sugars were released from keratinocytes and that UDP-glucose (UDP-Glc) added into keratinocyte cultures induced a specific, rapid induction of hyaluronan synthase 2 (HAS2), and an increase of hyaluronan synthesis. The up-regulation of HAS2 was associated with JAK2 and ERK1/2 activation, and specific Tyr(705) phosphorylation of transcription factor STAT3. Inhibition of JAK2, STAT3, or Gi-coupled receptors blocked the induction of HAS2 expression by UDP-Glc, the latter inhibitor suggesting that the signaling was triggered by the UDP-sugar receptor P2Y14. Chromatin immunoprecipitations demonstrated increased promoter binding of Tyr(P)(705)-STAT3 at the time of HAS2 induction. Interestingly, at the same time Ser(P)(727)-STAT3 binding to its response element regions in the HAS2 promoter was unchanged or decreased. UDP-Glc also stimulated keratinocyte migration, proliferation, and IL-8 expression, supporting a notion that UDP-Glc signals for epidermal inflammation, enhanced hyaluronan synthesis as an integral part of it.
Assuntos
Glucuronosiltransferase/genética , Ácido Hialurônico/biossíntese , Queratinócitos/metabolismo , Regiões Promotoras Genéticas , Receptores Purinérgicos P2/metabolismo , Fator de Transcrição STAT3/metabolismo , Tirosina/metabolismo , Uridina Difosfato Glucose/metabolismo , Motivos de Aminoácidos , Movimento Celular , Glucuronosiltransferase/metabolismo , Humanos , Hialuronan Sintases , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Queratinócitos/enzimologia , Fosforilação , Ligação Proteica , Receptores Purinérgicos P2/genética , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/genética , Transdução de Sinais , Tirosina/química , Tirosina/genética , Regulação para CimaRESUMO
Hyaluronan synthases (HAS1-3) are unique in that they are active only when located in the plasma membrane, where they extrude the growing hyaluronan (HA) directly into cell surface and extracellular space. Therefore, traffic of HAS to/from the plasma membrane is crucial for the synthesis of HA. In this study, we have identified Rab10 GTPase as the first protein known to be involved in the control of this traffic. Rab10 colocalized with HAS3 in intracellular vesicular structures and was co-immunoprecipitated with HAS3 from isolated endosomal vesicles. Rab10 silencing increased the plasma membrane residence of HAS3, resulting in a significant increase of HA secretion and an enlarged cell surface HA coat, whereas Rab10 overexpression suppressed HA synthesis. Rab10 silencing blocked the retrograde traffic of HAS3 from the plasma membrane to early endosomes. The cell surface HA coat impaired cell adhesion to type I collagen, as indicated by recovery of adhesion following hyaluronidase treatment. The data indicate a novel function for Rab10 in reducing cell surface HAS3, suppressing HA synthesis, and facilitating cell adhesion to type I collagen. These are processes important in tissue injury, inflammation, and malignant growth.
Assuntos
Colágeno Tipo I/metabolismo , Endocitose , Glucuronosiltransferase/metabolismo , Ácido Hialurônico/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cães , Glucuronosiltransferase/análise , Humanos , Hialuronan Sintases , Transporte Proteico , Interferência de RNA , Regulação para Cima , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.
Assuntos
Membrana Celular/metabolismo , Vesículas Revestidas/metabolismo , Ácido Hialurônico/metabolismo , Animais , Técnicas de Cultura de Células , Membrana Celular/ultraestrutura , Células Cultivadas , Cães , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hialuronan Sintases , Ácido Hialurônico/fisiologia , Masculino , Ratos , Ratos Wistar , TransfecçãoRESUMO
Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by â¼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed â¼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.
Assuntos
Acetilglucosamina/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/metabolismo , Difosfato de Uridina/química , Animais , Aorta/citologia , Células COS , Chlorocebus aethiops , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Glucosamina/metabolismo , Glucose/metabolismo , Humanos , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Inflamação , Isoenzimas , Modelos Biológicos , Neoplasias/enzimologiaRESUMO
Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested (â¼HA120 or â¼25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.
Assuntos
Membrana Celular/ultraestrutura , Citosol , Glucuronosiltransferase , Ácido Hialurônico , Sítios de Ligação , Citosol/química , Citosol/ultraestrutura , Endocitose , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Proteínas de Fluorescência Verde , Humanos , Hialuronan Sintases , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Células MCF-7 , Microinjeções , Oligossacarídeos/genéticaRESUMO
The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell-cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell-cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell-cell and cell-ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.
Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Glucuronosiltransferase/metabolismo , Ácido Hialurônico/biossíntese , Fuso Acromático/metabolismo , Animais , Adesão Celular/fisiologia , Técnicas de Cultura de Células , Linhagem Celular , Cães , Epitélio/metabolismo , Glucuronosiltransferase/genética , Hialuronan Sintases , Rim/citologia , Morfogênese/fisiologiaRESUMO
Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.
Assuntos
Epiderme/efeitos da radiação , Receptores de Hialuronatos/biossíntese , Ácido Hialurônico/biossíntese , Neoplasias Induzidas por Radiação/metabolismo , Lesões Pré-Cancerosas/metabolismo , Neoplasias Cutâneas/metabolismo , Raios Ultravioleta , Animais , Epiderme/metabolismo , Eritema/etiologia , Eritema/patologia , Feminino , Hiperplasia/metabolismo , Hiperplasia/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Neoplasias Induzidas por Radiação/patologia , Lesões Pré-Cancerosas/patologia , Neoplasias Cutâneas/patologiaRESUMO
Hyaluronan, a ubiquitous high-molecular-mass glycinoglycan on cell surfaces and in extracellular matrices, has a number of specific signaling functions in cell-cell communication. Changes in its content, molecular mass and turnover rate are crucial for cell proliferation, migration and apoptosis, processes that control tissue remodeling during embryonic development, inflammation, injury and cancer. To maintain tissue homeostasis, the synthesis of hyaluronan must therefore be tightly controlled. In this review, we highlight some recent data on the transcriptional regulation of hyaluronan synthase (Has1-3) expression and on the post-transcriptional control of hyaluronan synthase activity, which, in close association with the supply of the UDP-sugar substrates of hyaluronan synthase, adjust the rate of hyaluronan synthesis.
Assuntos
Ácido Hialurônico/biossíntese , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Animais , Desenvolvimento Embrionário , Homeostase , Humanos , Inflamação/metabolismo , Neoplasias/metabolismo , Ferimentos e Lesões/metabolismoRESUMO
BACKGROUND: Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity. METHODS: A total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry. RESULTS: The mRNA levels of HAS1-3 were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, HAS3 mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of HYAL1 mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. HYAL2 mRNA was also reduced in cancer (p = 0.02) and correlated with HYAL1 (r = 0.8, p = 0.0001). There was an inverse correlation between HYAL1 mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001). CONCLUSION: The results indicated that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for HASs were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.
Assuntos
Carcinoma Endometrioide/enzimologia , Moléculas de Adesão Celular/metabolismo , Neoplasias do Endométrio/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glucuronosiltransferase/metabolismo , Hialuronoglucosaminidase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Hialuronan Sintases , Pessoa de Meia-IdadeRESUMO
Hyaluronan synthases (HAS1-3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MbetaCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MbetaCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MbetaCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MbetaCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Glucuronosiltransferase/genética , Ácido Hialurônico/biossíntese , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , beta-Ciclodextrinas/farmacologia , Linhagem Celular Tumoral , Colesterol/metabolismo , Humanos , Hialuronan Sintases , Ácido Hialurônico/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The high-molecular-mass polysaccharide hyaluronan is abundant in the extracellular space between adjacent keratinocytes throughout the vital part of epidermis. It has a rapid turnover, and its content is subject to large fluctuations due to physiological and environmental conditions, with the strongest effects mediated by EGFR signaling. Using an elegant organotypic culture system, Monslow et al. (2009, this issue) demonstrate that heparin-binding (HB)-EGF released from its membrane anchor is the major ligand of EGFR in injured epidermis, accounting for the autocrine and paracrine activation of hyaluronan synthesis by the keratinocytes in the neighborhood, thus facilitating the epidermal wound-healing response.
Assuntos
Epiderme/lesões , Epiderme/metabolismo , Ácido Hialurônico/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Queratinócitos/metabolismo , Cicatrização , Animais , Células Epiteliais/fisiologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , HumanosRESUMO
BACKGROUND: Hyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1-3 immunoreactivity. METHODS: Normal ovaries (n = 5) and 34 serous epithelial ovarian tumors, divided into 4 groups: malignant grades 1+2 (n = 10); malignant grade 3 (n = 10); borderline (n = 4) and benign epithelial tumors (n = 10), were analyzed for mRNA by real-time RT-PCR and compared to hyaluronidase activity, hyaluronan staining, and HAS1-3 immunoreactivity in tissue sections of the same specimens. RESULTS: The levels of HAS2 and HAS3 mRNA (HAS1 was low or absent), were not consistently increased in the carcinomas, and were not significantly correlated with HAS protein or hyaluronan accumulation in individual samples. Instead, the median of HYAL1 mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of HYAL1, but not HYAL2, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between HYAL1 mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025). CONCLUSION: The results indicate that in serous epithelial ovarian malignancies HAS expression is not consistently elevated but HYAL1 expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words).
Assuntos
Carcinoma/enzimologia , Regulação Neoplásica da Expressão Gênica , Glucuronosiltransferase/genética , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/genética , Neoplasias Ovarianas/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Feminino , Glucuronosiltransferase/metabolismo , Humanos , Hialuronan Sintases , Hialuronoglucosaminidase/metabolismo , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Adulto JovemRESUMO
Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.
Assuntos
Glucuronosiltransferase/metabolismo , Ácido Hialurônico/biossíntese , Himecromona/análogos & derivados , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Glucuronosiltransferase/genética , Humanos , Hialuronan Sintases , Himecromona/farmacologia , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Invasividade Neoplásica/prevenção & controle , Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
Hyaluronan attached to cell surface can form at least two very different structures; a pericellular coat close to plasma membrane and hyaluronan chains coalesced into "cables" that can span several cell lengths. The hyaluronan in cables, induced by many inflammatory agents, can bind leukocytes, whereas that in the pericellular coat does not contribute to leukocyte binding. Therefore, this structural change seems to have a major role in inflammation. In the present study we checked whether cells of squamous epithelium, like epidermal keratinocytes, can form hyaluronan cables and bind leukocytes. In addition, we checked whether hyaluronan synthesis is affected during the induction of cables. Control keratinocytes expressed pericellular hyaluronan as small patches on plasma membrane. But when treated with inflammatory agents or stressful conditions (tunicamycin, interleukin-1beta, tumor necrosis factor-alpha, and high glucose concentration), hyaluronan organization changed into cable-like structures that avidly bound monocytes. Simultaneously, the total amount of secreted hyaluronan was slightly decreased, and the expression levels of hyaluronan synthases (Has1-3) and CD44 were not significantly changed. The results show that epidermal keratinocytes can form cables and bind leukocytes under inflammatory provocation and that these effects are not dependent on stimulation of hyaluronan secretion.