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1.
Neurochem Res ; 45(1): 68-82, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31664654

RESUMO

Hapln4 is a link protein which stabilizes the binding between lecticans and hyaluronan in perineuronal nets (PNNs) in specific brain regions, including the medial nucleus of the trapezoid body (MNTB). The aim of this study was: (1) to reveal possible age-related alterations in the extracellular matrix composition in the MNTB and inferior colliculus, which was devoid of Hapln4 and served as a negative control, (2) to determine the impact of the Hapln4 deletion on the values of the ECS diffusion parameters in young and aged animals and (3) to verify that PNNs moderate age-related changes in the ECS diffusion, and that Hapln4-brevican complex is indispensable for the correct protective function of the PNNs. To achieve this, we evaluated the ECS diffusion parameters using the real-time iontophoretic method in the selected region in young adult (3 to 6-months-old) and aged (12 to 18-months-old) wild type and Hapln4 knock-out (KO) mice. The results were correlated with an immunohistochemical analysis of the ECM composition and astrocyte morphology. We report that the ECM composition is altered in the aged MNTB and aging is a critical point, revealing the effect of Hapln4 deficiency on the ECS diffusion. All of our findings support the hypothesis that the ECM changes in the MNTB of aged KO animals affect the ECS parameters indirectly, via morphological changes of astrocytes, which are in direct contact with synapses and can be influenced by the ongoing synaptic transmission altered by shifts in the ECM composition.

2.
Adv Exp Med Biol ; 1190: 107-122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31760641

RESUMO

Enriched Na+ channel clustering allows for rapid saltatory conduction at a specialized structure in myelinated axons, the node of Ranvier, where cations are exchanged across the axon membrane. In the extracellular matrix (ECM), highly negatively charged molecules accumulate and wrap around the nodal gaps creating an ECM dome, called the perinodal ECM. The perinodal ECM has different molecular compositions in the central nervous system (CNS) and peripheral nervous system (PNS). Chondroitin sulfate proteoglycans are abundant in the ECM at the CNS nodes, whereas heparan sulfate proteoglycans are abundant at the PNS nodes. The proteoglycans have glycosaminoglycan chains on their core proteins, which makes them electrostatically negative. They associate with other ECM molecules and form a huge stable ECM complex at the nodal gaps. The polyanionic molecular complexes have high affinity to cations and potentially contribute to preventing cation diffusion at the nodes.In this chapter, we describe the molecular composition of the perinodal ECM in the CNS and PNS, and discuss their physiological role at the node of Ranvier.


Assuntos
Sistema Nervoso Central/fisiologia , Matriz Extracelular/fisiologia , Sistema Nervoso Periférico/fisiologia , Nós Neurofibrosos/fisiologia , Axônios/fisiologia , Sulfatos de Condroitina/fisiologia , Glicosaminoglicanos/fisiologia , Heparitina Sulfato/fisiologia , Humanos , Proteoglicanas/fisiologia
3.
Cells Tissues Organs ; 207(3-4): 115-126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31574516

RESUMO

Stem cells have essential applications in in vitro tissue engineering or regenerative medicine. However, there is still a need to understand more deeply the mechanisms of stem cell differentiation and to optimize the methods to control stem cell function. In this study, we first investigated the activity of DNA methyltransferases (DNMTs) during chondrogenic differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (hBMSCs) and found that DNMT3A and DNMT3B were markedly upregulated during hBMSC chondrogenic differentiation. In an attempt to understand the effect of DNMT3A and DNMT3B on the chondrogenic differentiation of hBMSCs, we transiently transfected the cells with expression vectors for the two enzymes. Interestingly, DNMT3A overexpression strongly enhanced the chondrogenesis of hBMSCs, by increasing the gene expression of the mature chondrocyte marker, collagen type II, more than 200-fold. Analysis of the methylation condition in the cells revealed that DNMT3A and DNMT3B methylated the promoter sequence of early stem cell markers, NANOG and POU5F1(OCT-4). Conversely, the suppression of chondrogenic differentiation and the increase in stem cell markers of hBMSCs were obtained by chemical stimulation with the demethylating agent, 5-azacitidine. Loss-of-function assays with siRNAs targeting DNMT3A also significantly suppressed the chondrogenic differentiation of hBMSCs. Together, these results not only show the critical roles of DNMTs in regulating the chondrogenic differentiation of hBMSCs, but also suggest that manipulation of DNMT activity can be important tools to enhance the differentiation of hBMSCs towards chondrogenesis for potential application in cartilage tissue engineering or cartilage regeneration.

4.
Int J Mol Sci ; 20(19)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554264

RESUMO

Epithelial keratinization involves complex cellular modifications that provide protection against pathogens and chemical and mechanical injuries. In the oral cavity, keratinized mucosa is also crucial to maintain healthy periodontal or peri-implant tissues. In this study, we investigated the roles of type XVIII collagen, a collagen-glycosaminoglycan featuring an extracellular matrix component present in the basement membrane, in oral mucosal keratinization. Histological analysis of keratinized and non-keratinized oral mucosa showed that type XVIII collagen was highly expressed in keratinized mucosa. Additionally, a 3D culture system using human squamous carcinoma cells (TR146) was used to evaluate and correlate the changes in the expression of type XVIII collagen gene, COL18A1, and epithelial keratinization-related markers, e.g., keratin 1 (KRT1) and 10 (KRT10). The results showed that the increase in COL18A1 expression followed the increase in KRT1 and KRT10 mRNA levels. Additionally, loss-of-function analyses using silencing RNA targeting COL18A1 mRNA and a Col18-knockout (KO) mouse revealed that the absence of type XVIII collagen induces a dramatic decrease in KRT10 expression as well as in the number and size of keratohyalin granules. Together, the results of this study demonstrate the importance of type XVIII collagen in oral mucosal keratinization.


Assuntos
Colágeno Tipo XVIII/metabolismo , Grânulos Citoplasmáticos/metabolismo , Queratinas/metabolismo , Mucosa Bucal/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Colágeno Tipo XVIII/genética , Imunofluorescência , Humanos , Camundongos , Camundongos Knockout
5.
Commun Biol ; 2: 346, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31552299

RESUMO

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1 flox/flox and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system.


Assuntos
Sistema A de Transporte de Aminoácidos/antagonistas & inibidores , Sistema A de Transporte de Aminoácidos/metabolismo , Autofagia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Sistema A de Transporte de Aminoácidos/genética , Animais , Infarto Cerebral/etiologia , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Expressão Gênica , Loci Gênicos , Genoma , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuroproteção , Especificidade de Órgãos
6.
Exp Cell Res ; 383(2): 111556, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31415758

RESUMO

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1ß and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

7.
Nat Rev Neurosci ; 20(8): 451-465, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31263252

RESUMO

Perineuronal nets (PNNs) are extracellular matrix (ECM) chondroitin sulfate proteoglycan (CSPG)-containing structures that surround the soma and dendrites of various mammalian neuronal cell types. PNNs appear during development around the time that the critical periods for developmental plasticity end and are important for both their onset and closure. A similar structure - the perinodal ECM - surrounds the axonal nodes of Ranvier and appears as myelination is completed, acting as an ion-diffusion barrier that affects axonal conduction speed. Recent work has revealed the importance of PNNs in controlling plasticity in the CNS. Digestion, blocking or removal of PNNs influences functional recovery after a variety of CNS lesions. PNNs have further been shown to be involved in the regulation of memory and have been implicated in a number of psychiatric disorders.


Assuntos
Matriz Extracelular/fisiologia , Neurônios/fisiologia , Animais , Doenças do Sistema Nervoso Central/fisiopatologia , Humanos , Memória/fisiologia , Transtornos Mentais/fisiopatologia , Modelos Neurológicos , Plasticidade Neuronal/fisiologia
8.
Biochem Biophys Res Commun ; 516(4): 1229-1233, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31300199

RESUMO

Global gene deletion studies have established that Runt-related transcription factor-2 (Runx2) is essential during skeletogenesis for osteoblastic differentiation in both intramembranous and endochondral ossification processes. However, the postnatal significance of Runx2 in vivo is poorly understood because a global Runx2 deletion causes perinatal lethality. In this study, we generated tamoxifen-induced Runx2 global deficient mice by crossing Runx2flox mice with ROSA26-CreERT2 mice (Rosa26-CreERT2; Runx2flox/flox). Four-week-old mice were intraperitoneally treated with tamoxifen for five consecutive days, sacrificed, and analyzed six weeks after tamoxifen administration. Deletion of Runx2 led to low bone mass, which is associated with decreased bone formation and bone resorption as well as excessive bone marrow adiposity. Collectively, postnatal Runx2 absolutely plays an important role in maintaining the homeostasis of bone tissues not only in bone mass, but also in the bone marrow environment.

9.
Acta Med Okayama ; 73(2): 135-146, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31015748

RESUMO

The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization.


Assuntos
Membrana Basal/fisiologia , Colágeno Tipo XVIII/metabolismo , Células Epidérmicas/metabolismo , Cicatrização/fisiologia , Animais , Membrana Basal/ultraestrutura , Epiderme/patologia , Junções Intercelulares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real/métodos
10.
Int J Mol Sci ; 20(5)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836626

RESUMO

A deeper understanding of the detailed mechanism of in vivo tissue healing is necessary for the development of novel regenerative therapies. Among several external factors, environmental pH is one of the crucial parameters that greatly affects enzyme activity and cellular biochemical reactions involving tissue repair and homeostasis. In this study, in order to analyze the microenvironmental conditions during bone healing, we first measured the pH in vivo at the bone healing site using a high-resolution fiber optic pH microsensor directly in femur defects and tooth extraction sockets. The pH was shown to decrease from physiological 7.4 to 6.8 during the initial two days of healing (inflammatory phase). In the same initial stages of the inflammatory phase of the bone healing process, mesenchymal stem cells (MSCs) are known to migrate to the healing site to contribute to tissue repair. Therefore, we investigated the effect of a short-term acidic (pH 6.8) pre-treatment on the stemness of bone marrow-derived MSCs (BMSCs). Interestingly, the results showed that pre-treatment of BMSCs with acidic pH enhances the expression of stem cell markers (OCT-4, NANOG, SSEA-4), as well as cell viability and proliferation. On the other hand, acidic pH decreased BMSC migration ability. These results indicate that acidic pH during the initial stages of bone healing is important to enhance the stem cell properties of BMSCs. These findings may enable the development of novel methods for optimization of stem cell function towards tissue engineering or regenerative medicine.


Assuntos
Ácidos/farmacologia , Regeneração Óssea/genética , Osteogênese/efeitos dos fármacos , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Medicina Regenerativa , Antígenos Embrionários Estágio-Específicos/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Cicatrização/genética
11.
J Bone Miner Res ; 34(2): 327-332, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30352125

RESUMO

Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.

12.
J Cosmet Dermatol ; 2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30548159

RESUMO

INTRODUCTION: Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70 years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. METHODS: Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. RESULTS: In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. CONCLUSION: Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.

13.
J Orthop Res ; 36(12): 3247-3255, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30117186

RESUMO

Hyaluronan (HA) is an extracellular matrix (ECM) component of articular cartilage and has been used to treat patients with osteoarthritis (OA). A disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) play an important role in cartilage degradation in OA. We have previously reported that ADAMTS4 and ADAMTS9 were induced by cytokine stimulation. However, the effect of HA on the cytokine-inducible ADAMTS9 has never been investigated. Moreover, it is unclear whether HA protects cartilage by suppressing aggrecan degradation. Here, we examined the effects of HA on ADAMTS expression in vitro and on cartilage degradation in vivo. ADAMTS9 expression was higher than that of the other aggrecanases (ADAMTS4 and 5) in human chondrocytes, chondrocytic cells, and rat cartilage. ADAMTS4 and 9 mRNA levels were upregulated in cytokine-stimulated chondrocytes and chondrocytic cells. Pre-incubation with HA significantly inhibited ADAMTS9 mRNA expression in cytokine-stimulated cells. In a rat OA model, Adamts5 and 9 mRNA levels were transiently increased after surgery; intra-articular HA injections attenuated the induction of Adamts5 and 9 mRNA. HA also blocked aggrecan cleavage by aggrecanase in OA rats in a molecular size-dependent manner. These results demonstrate that HA attenuates induced aggrecanases expression in OA and thereby protects articular cartilage degradation by this enzyme. Our findings provide insight into the molecular basis for the beneficial effects of HA in OA. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:3247-3255, 2018.


Assuntos
Cartilagem Articular/efeitos dos fármacos , Endopeptidases/genética , Ácido Hialurônico/farmacologia , Proteína ADAMTS5/genética , Proteína ADAMTS9/genética , Agrecanas/metabolismo , Animais , Cartilagem Articular/metabolismo , Células Cultivadas , Humanos , Receptores de Hialuronatos/fisiologia , Masculino , Peso Molecular , Osteoartrite/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley
14.
J Neurochem ; 147(6): 748-763, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30125937

RESUMO

Purkinje cells (PCs) convey the sole output of the cerebellar cortex to the deep cerebellar nuclei (DCN). DCN neurons are enwrapped in densely organized extracellular matrix structures, known as perineuronal nets (PNNs). PNNs are typically found around fast-spiking GABAergic interneurons expressing parvalbumin but interestingly also exist surrounding other neurons, such as the neurons in the DCN and medial nucleus of the trapezoid body, which are the post-synaptic neurons of large axo-somatic synapses adapted for fast signaling. This characteristic localization prompted the hypothesis that PNNs might play a role in the maintenance and formation of large fast-signaling synapses. To elucidate the role of the PNN at these synapses, we investigated the electrophysiological and morphological properties of DCN synapses in hyaluronan and proteoglycan binding link protein 4 (Hapln4/Bral2) knockout (KO) mice around postnatal day (P)14. Hapln4/Bral2 is important for PNN structure, as it stabilizes the interaction between hyaluronan and proteoglycan. Here, using immunohistochemistry we show that Hapln4/Bral2 localized closely with GABAergic terminals. In DCN neurons of Hapln4/Bral2 KO mice, inhibitory synaptic strengths were reduced as compared to those in wild-type mice, whereas the properties of excitatory synapses were unaffected. The reduced IPSC amplitudes were mainly because of reduced numbers of releasable vesicles. Moreover, Hapln4/Bral2 deficiency reduced the number of PC GABAergic terminals in the DCN. These results demonstrate that Hapln4/Bral2 is a PNN component that selectively contributes to formation and transmission of PC-DCN synapses in the cerebellum. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.


Assuntos
Cerebelo/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Células de Purkinje/fisiologia , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Cerebelo/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteínas da Matriz Extracelular/biossíntese , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Técnicas de Patch-Clamp
15.
Acta Med Okayama ; 72(3): 257-266, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29926003

RESUMO

Several research groups demonstrated that 'a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS)'-family proteases play roles in cancer progression. However, the origins and contributions of these proteases are not known. Here, we demonstrate an association between host-produced ADAMTS4 and early-stage tumor growth. Murine Lewis lung carcinoma (LLC) tumors showed marked expressions of Adamts4 and Adamts5. We examined the contributions and distributions of host-derived Adamts4 and Adamts5 on tumor growth, using Adamts4LacZ/LacZ and Adamts5LacZ/LacZ knockout mice. Interestingly, the Adamts4LacZ/LacZ mice showed enhanced tumor growth compared to wild-type mice at 5-, 10- and 12-days post-inoculation, whereas the Adamts5LacZ/LacZ mice did not show significant differences in tumor growth. We next examined LacZ distribution in LLC tumor-bearing Adamts4LacZ/LacZ mice by ß-galactosidase (ß-gal) staining. We found that the ß-gal-positive signals were strictly localized at the interior areas of the tumor at 10 days post-inoculation. Multiple staining demonstrated that most of the ß-gal-positive cells were localized at the tumor vasculature in Adamts4LacZ/LacZ mice. Interestingly, ß-gal-positive signals were not co-localized with biglycan after 10 days post-inoculation, excluding the biglycan cleavage by host-derived ADAMTS4. Taken together, these findings illustrate that host-derived ADAMTS4 was expressed at the tumor vessels and was associated with early-stage tumor growth.


Assuntos
Proteína ADAMTS4/fisiologia , Neoplasias Experimentais/patologia , Proteína ADAMTS4/análise , Proteína ADAMTS5/análise , Proteína ADAMTS5/fisiologia , Animais , Proliferação de Células , Células Endoteliais/química , Camundongos , Camundongos Endogâmicos C57BL , Estadiamento de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/prevenção & controle , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise
16.
Sci Rep ; 8(1): 2612, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29422532

RESUMO

Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization. These findings indicate that α112:α556 (IV) network, which is the only network that includes the α6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.


Assuntos
Membrana Basal/metabolismo , Colágeno Tipo IV/fisiologia , Queratina-10/metabolismo , Queratinócitos/fisiologia , Mucosa Bucal/metabolismo , Animais , Diferenciação Celular , Colágeno Tipo IV/genética , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL
17.
J Cell Physiol ; 233(6): 4825-4840, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29150954

RESUMO

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates.


Assuntos
Condrócitos/metabolismo , Condrogênese , Osteoblastos/metabolismo , Osteogênese , RNA Longo não Codificante/metabolismo , Células-Tronco/metabolismo , Animais , Desdiferenciação Celular , Linhagem Celular Tumoral , Senescência Celular , Condrogênese/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Osteogênese/genética , Fenótipo , Primatas , RNA Longo não Codificante/genética , Transdução de Sinais
18.
J Neurosci Res ; 96(2): 313-327, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28815777

RESUMO

Bral2 is a link protein stabilizing the binding between lecticans and hyaluronan in perineuronal nets and axonal coats (ACs) in specific brain regions. Using the real-time iontophoretic method and diffusion-weighted magnetic resonance, we determined the extracellular space (ECS) volume fraction (α), tortuosity (λ), and apparent diffusion coefficient of water (ADCW ) in the thalamic ventral posteromedial nucleus (VPM) and sensorimotor cortex of young adult (3-6 months) and aged (14-20 months) Bral2-deficient (Bral2-/- ) mice and age-matched wild-type (wt) controls. The results were correlated with an analysis of extracellular matrix composition. In the cortex, no changes between wt and Bral2-/- were detected, either in the young or aged mice. In the VPM of aged but not in young Bral2-/- mice, we observed a significant decrease in α and ADCW in comparison with age-matched controls. Bral2 deficiency led to a reduction of both aggrecan- and brevican-associated perineuronal nets and a complete disruption of brevican-based ACs in young as well as aged VPM. Our data suggest that aging is a critical point that reveals the effect of Bral2 deficiency on VPM diffusion. This effect is probably mediated through the enhanced age-related damage of neurons lacking protective ACs, or the exhausting of compensatory mechanisms maintaining unchanged diffusion parameters in young Bral2-/- animals. A decreased ECS volume in aged Bral2-/- mice may influence the diffusion of neuroactive substances, and thus extrasynaptic and also indirectly synaptic transmission in this important nucleus of the somatosensory pathway.


Assuntos
Envelhecimento/fisiologia , Proteínas da Matriz Extracelular/deficiência , Espaço Extracelular/genética , Proteínas do Tecido Nervoso/deficiência , Neurônios/citologia , Tálamo/citologia , Agrecanas/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Imagem de Difusão por Ressonância Magnética , Proteínas da Matriz Extracelular/genética , Espaço Extracelular/diagnóstico por imagem , Feminino , Estimulantes Ganglionares/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , RNA Mensageiro
19.
Sci Rep ; 7(1): 17225, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29222454

RESUMO

The proteoglycan versican is implicated in growth and metastases of several cancers. Here we investigated a potential contribution of stromal versican to tumor growth and angiogenesis. We initially determined versican expression by several cancer cell lines. Among these, MDA-MB231 and B16F10 had none to minimal expression in contrast to Lewis lung carcinoma (LLC). Notably, tumors arising from these cell lines had higher versican levels than the cell lines themselves suggesting a contribution from the host-derived tumor stroma. In LLC-derived tumors, both the tumor and stroma expressed versican at high levels. Thus, tumor stroma can make a significant contribution to tumor versican content. Versican localized preferentially to the vicinity of tumor vasculature and macrophages in the tumor. However, an ADAMTS protease-generated versican fragment uniquely localized to vascular endothelium. To specifically determine the impact of host/stroma-derived versican we therefore compared growth of tumors from B16F10 cells, which produced littleversican, in Vcan hdf/+ mice and wild-type littermates. Tumors in Vcan hdf/+ mice had reduced growth with a lower capillary density and accumulation of capillaries at the tumor periphery. These findings illustrate the variability of tumor cell line expression of versican, and demonstrate that versican is consistently contributed by the stromal tissue, where it contributes to tumor angiogenesis.


Assuntos
Neovascularização Patológica/metabolismo , Células Estromais/metabolismo , Versicanas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Macrófagos/metabolismo , Camundongos , Metástase Neoplásica , Proteólise , Microambiente Tumoral , Versicanas/biossíntese , Versicanas/genética
20.
Acta Histochem Cytochem ; 50(2): 71-84, 2017 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-28522882

RESUMO

We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.

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