Correlation of syndecan gene amplification with metastatic potential and clinical outcomes in carcinomas.

Cell surface receptors play crucial roles in cellular responses to extracellular ligands, helping to modulate the functions of a cell based on information coming from outside the cell. Syndecan refers to a family of cell adhesion receptors that regulate both extracellular and cytosolic events. Alteration of syndecan expression disrupts regulatory mechanisms in a cell type-specific fashion, often leading to serious diseases, notably cancer. Given the multifaceted functions and distinct tissue distributions of syndecan, it will be important to unravel the gene-level intricacies of syndecan expression and thereby further understand its involvement in various carcinogenic processes. Although accumulating evidence indicates that the protein expression patterns of syndecan family members are significantly altered in cancer cells, the underlying gene-level mechanisms remain largely unknown. This review endeavors to explore syndecan gene expression levels across different cancer types by scrutinizing extensive cancer genome datasets utilizing tools such as cBioPortal. Our analysis unveils that somatic mutations in SDC genes are rare occurrences, whereas copy number alterations are frequently observed across diverse cancers, particularly in SDC2 and SDC4. Notably, amplifications of SDC2 and SDC4 correlate with heightened metastatic potential and dismal prognosis. This underscores the recurrent nature of SDC2 and SDC4 amplifications during carcinogenesis and sheds light on their role in promoting cancer activity through augmented protein expression. The identification of these amplifications not only enriches our understanding of carcinogenic mechanisms but also hints at the potential therapeutic avenue of targeting SDC2 and SDC4 to curb cancer cell proliferation and metastasis.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.

Hexosamine biosynthetic pathway and O-GlcNAc cycling of glucose metabolism in brain function and disease.

Impaired brain glucose metabolism is considered a hallmark of brain dysfunction and neurodegeneration. Disruption of the hexosamine biosynthetic pathway (HBP) and subsequent O-linked N-acetylglucosamine (O-GlcNAc) cycling has been identified as an emerging link between altered glucose metabolism and defects in the brain. Myriads of cytosolic and nuclear proteins in the nervous system are modified at serine or threonine residues with a single N-acetylglucosamine (O-GlcNAc) molecule by O-GlcNAc transferase (OGT), which can be removed by ß-N-acetylglucosaminidase (O-GlcNAcase, OGA). Homeostatic regulation of O-GlcNAc cycling is important for the maintenance of normal brain activity. Although significant evidence linking dysregulated HBP metabolism and aberrant O-GlcNAc cycling to induction or progression of neuronal diseases has been obtained, the issue of whether altered O-GlcNAcylation is causal in brain pathogenesis remains uncertain. Elucidation of the specific functions and regulatory mechanisms of individual O-GlcNAcylated neuronal proteins in both normal and diseased states may facilitate the identification of novel therapeutic targets for various neuronal disorders. The information presented in this review highlights the importance of HBP/O-GlcNAcylation in the neuronal system and summarizes the roles and potential mechanisms of O-GlcNAcylated neuronal proteins in maintaining normal brain function and initiation and progression of neurological diseases.

Repeated sleep deprivation decreases the flux into hexosamine biosynthetic pathway/O-GlcNAc cycling and aggravates Alzheimer's disease neuropathology in adult zebrafish.

This study investigated chronic and repeated sleep deprivation (RSD)-induced neuronal changes in hexosamine biosynthetic pathway/O-linked N-acetylglucosamine (HBP/O-GlcNAc) cycling of glucose metabolism and further explored the role of altered O-GlcNAc cycling in promoting neurodegeneration using an adult zebrafish model. RSD-triggered degenerative changes in the brain led to impairment of memory, neuroinflammation and amyloid beta (Aß) accumulation. Metabolite profiling of RSD zebrafish brain revealed a significant decrease in glucose, indicating a potential association between RSD-induced neurodegeneration and dysregulated glucose metabolism. While RSD had no impact on overall O-GlcNAcylation levels in the hippocampus region, changes were observed in two O-GlcNAcylation-regulating enzymes, specifically, a decrease in O-GlcNAc transferase (OGT) and an increase in O-GlcNAcase (OGA). Glucosamine (GlcN) treatment induced an increase in O-GlcNAcylation and recovery of the OGT level that was decreased in the RSD group. In addition, GlcN reversed cognitive impairment by RSD. GlcN reduced neuroinflammation and attenuated Aß accumulation induced by RSD. Repeated treatment of zebrafish with diazo-5-oxo-l-norleucine (DON), an inhibitor of HBP metabolism, resulted in cognitive dysfunction, neuroinflammation and Aß accumulation, similar to the effects of RSD. The pathological changes induced by DON were restored to normal upon treatment with GlcN. Both the SD and DON-treated groups exhibited a common decrease in glutamate and γ-aminobutyric acid compared to the control group. Overexpression of OGT in zebrafish brain rescued RSD-induced neuronal dysfunction and neurodegeneration. RSD induced a decrease in O-GlcNAcylation of amyloid precursor protein and increase in ß-secretase activity, which were reversed by GlcN treatment. Based on the collective findings, we propose that dysregulation of HBP and O-GlcNAc cycling in brain plays a crucial role in RSD-mediated progression of neurodegeneration and Alzheimer's disease pathogenesis. Targeting of this pathway may, therefore, offer an effective regulatory approach for treatment of sleep-associated neurodegenerative disorders.

Sleep deprivation impairs learning and memory by decreasing protein O-GlcNAcylation in the brain of adult zebrafish.

Sleep is an evolutionarily conserved physiological process implicated in the consolidation of learning and memory (L/M). Here, we report that sleep deprivation (SD)-induced cognitive deficits in zebrafish are mediated through reduction in O-GlcNAcylation of brain. Microarray-based gene expression profiling of zebrafish brain demonstrated significant changes in genes involved in metabolism by SD or fear conditioning (FC), compared to the control group. In particular, it was observed that a marked decrease in the number of genes involved in carboxylic acid and organic acid metabolic processes in the brains of SD group compared to control group. SD downregulated O-GlcNAc transferase (OGT) and O-GlcNAcylation, while the expression of O-GlcNAcase was upregulated. FC activated protein kinase A (PKA) and phosphorylated cAMP response element binding protein (p-CREB), an effect that was greatly inhibited by SD. Moreover, FC upregulated expressions of OGT and increased O-GlcNAcylation in the brains of normal but not SD zebrafish. Intriguingly, upregulation of O-GlcNAcylation by glucosamine restored defects in L/M functions and PKA/p-CREB activity in SD group. Our findings highlight the O-GlcNAcylation changes in the brain during the L/M process and further provide a foundation for future studies seeking the molecular and biochemical mechanisms by which HBP of glucose metabolism affects cognitive function.

Glucosamine improves survival in a mouse model of sepsis and attenuates sepsis-induced lung injury and inflammation.

The aim of the current study was to investigate the effects of glucosamine (GlcN) on septic lethality and sepsis-induced inflammation using animal models of mice and zebrafish. GlcN pretreatment improved survival in the cecal ligation and puncture (CLP)-induced sepsis mouse model and attenuated lipopolysaccharide (LPS)-induced septic lung injury and systemic inflammation. GlcN suppressed LPS-induced M1-specific but not M2-specific gene expression. Furthermore, increased expressions of inflammatory genes in visceral tissue of LPS-injected zebrafish were suppressed by GlcN. GlcN suppressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and NF-κB in lung tissue. LPS triggered a reduction in O-GlcNAc levels in nucleocytoplasmic proteins of lung, liver, and spleen after 1 day, which returned to normal levels at day 3. GlcN inhibited LPS-induced O-GlcNAc down-regulation in mouse lung and visceral tissue of zebrafish. Furthermore, the O-GlcNAcase (OGA) level was increased by LPS, which were suppressed by GlcN in mouse and zebrafish. OGA inhibitors suppressed LPS-induced expression of inflammatory genes in RAW264.7 cells and the visceral tissue of zebrafish. Stable knockdown of Oga via short hairpin RNA led to increased inducible nitric oxide synthase (iNOS) expression in response to LPS with or without GlcN in RAW264.7 cells. Overall, our results demonstrate a protective effect of GlcN on sepsis potentially through modulation of O-GlcNAcylation of nucleocytoplasmic proteins.

A caffeic acid-ferulic acid hybrid compound attenuates lipopolysaccharide-mediated inflammation in BV2 and RAW264.7 cells.

In the present study, we synthesized and evaluated the anti-inflammatory effects of the two component hybrids, caffeic acid (CA)-ferulic acid (FA), FA-Tryptamine (Trm), CA-Piperonyl Triazol (PT) and FA-PT. Of these five hybrids, CA-FA had the most potent inhibitory effect on butyrylcholinesterase (BuChE) activity. The CA containing hybrids, CA-FA, CA-Trm, and CA-PT, dose-dependently inhibited LPS-induced nitric oxide (NO) generation in BV2 cells, whereas FA-PT, FA-Trm, CA, FA, Trm, and PT did not. Although CA-FA, CA-Trm and CA-PT had similar inhibitory effects on LPS-induced NO generation, CA-FA best protected BV2 cells from LPS-induced cell death. CA-FA, but not CA or FA, dose-dependently inhibited LPS-induced up-regulations of NO synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions in BV2 and RAW264.7 cells. Furthermore, CA-FA inhibited LPS-induced iNOS, COX-2, interleukin-6, and interleukin-1ß mRNA expressions in BV2 cells. CA-FA also inhibited the LPS-induced phosphorylations of STAT3, Akt, and IκB and selectively inhibited LPS-induced NF-κB activation. Overall, our data suggest that CA-FA has BuChE inhibitory effects and down-regulates inflammatory responses by inhibiting NF-κB, which indicates CA-FA be viewed as a potential therapeutic agent for the treatment of inflammatory diseases of the peripheral system and central nervous systems.

Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells.

We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) under various glucose conditions in macrophage cells. At normal glucose concentrations, GlcN dose dependently increased LPS-stimulated production of NO/iNOS. However, GlcN suppressed NO/iNOS production under high glucose culture conditions. Moreover, GlcN suppressed LPS-induced up-regulation of COX-2, IL-6, and TNF-α mRNAs under 25 mm glucose conditions yet did not inhibit up-regulation under 5 mm glucose conditions. Glucose itself dose dependently increased LPS-induced iNOS expression. LPS-induced MAPK and IκB-α phosphorylation did not significantly differ at normal and high glucose conditions. The activity of LPS-induced nuclear factor-κB (NF-κB) and DNA binding of c-Rel to the iNOS promoter were inhibited under high glucose conditions in comparison with no significant changes under normal glucose conditions. In addition, we found that the LPS-induced increase in O-GlcNAcylation as well as DNA binding of c-Rel to the iNOS promoter were further increased by GlcN under normal glucose conditions. However, both O-GlcNAcylation and DNA binding of c-Rel decreased under high glucose conditions. The NF-κB inhibitor, pyrrolidine dithiocarbamate, inhibited LPS-induced iNOS expression under high glucose conditions but it did not influence iNOS induction under normal glucose conditions. In addition, pyrrolidine dithiocarbamate inhibited NF-κB DNA binding and c-Rel O-GlcNAcylation only under high glucose conditions. By blocking transcription with actinomycin D, we found that stability of LPS-induced iNOS mRNA was increased by GlcN under normal glucose conditions. These results suggest that GlcN regulates inflammation by sensing energy states of normal and fuel excess.

A unique phenylalanine in the transmembrane domain strengthens homodimerization of the syndecan-2 transmembrane domain and functionally regulates syndecan-2.

The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe(167)) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe(167) was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe(167) in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members.

Exogenous Sodium Pyruvate Stimulates Adipogenesis of 3T3-L1 Cells.

We investigated the effects of exogenous sodium pyruvate (SP) on adipocyte differentiation, lipid accumulation, and the mRNA expression levels of adipogenesis-related genes in 3T3-L1 pre-adipocytes. Differentiation of pre-adipocytes was induced by MDI (3-isobutyl-1-methylxanthine: IBMX, dexamethasone: DEX, and insulin), in the presence or absence of SP. Adipogenesis was stimulated by SP in a concentration-dependent manner. SP also induced the expression of genes encoding aP2, GLUT4, and adiponectin, but had no effect on cell proliferation. Exogenous glucose did not promote adipogenesis or lipid accumulation. 2-deoxy-D-glucose inhibited adipogenesis initiated by MDI, but failed to influence the effects of SP on adipogenesis, whereas 3-bromopyruvate inhibited adipogenesis regardless of whether SP was present. The pro-adipogenic properties of SP were limited to the early events of adipogenesis. To determine whether SP mimics the adipogenic action of dexamethasone or insulin, we examined the effects of SP on adipogenesis with combinations of IBMX, DEX, and insulin. SP did not improve incomplete lipid accumulation observed in cells grown under IBMX-, DEX-, or insulin-free conditions. Insulin-stimulated ERK1/2 phosphorylation was diminished by SP, while phosphorylation of Akt was increased, correlating with increased glucose uptake in response to insulin. We also observed that SP stimulated immediate early expression of C/EBPß and C/EBPδ. The PPARγ antagonist GW9662 inhibited adipogenesis. Our findings highlight the adipogenic function of exogenous SP by stimulating early events of adipogenesis.

Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

Glucosamine increases macrophage lipid accumulation by regulating the mammalian target of rapamycin signaling pathway.

Elevated blood glucose is associated with an increased risk of atherosclerosis. Data from the current study showed that glucosamine (GlcN), a normal glucose metabolite of the hexosamine biosynthetic pathway (HBP), promoted lipid accumulation in RAW264.7 macrophage cells. Oleic acid- and lipopolysaccharide (LPS)-induced lipid accumulation was further enhanced by GlcN in RAW264.7 cells, although there was no a significant change in the rate of fatty acid uptake. GlcN increased acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), scavenger receptor class A, liver X receptor, and sterol regulatory elementbinding protein-1c (SREBP-1c) mRNA expression, and; conversely, suppressed ATP-binding cassette transporter A1 (ABCA-1) and ABCG-1 expression. Additionally, GlcN promoted O-GlcNAcylation of nuclear SREBP-1 but did not affect its DNA binding activity. GlcN stimulated phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Rapamycin, a mTOR-specific inhibitor, suppressed GlcN-induced lipid accumulation in RAW264.7 cells. The GlcN-mediated increase in ACC and FAS mRNA was suppressed, while the decrease in ABCA-1 and ABCG-1 by GlcN was not significantly altered by rapamycin. Together, our results highlight the importance of the mTOR signaling pathway in GlcN-induced macrophage lipid accumulation and further support a potential link between mTOR and HBP signaling in lipogenesis. [BMB Reports 2024; 57(2): 92-97].

Forskolin rescues hypoxia-induced cognitive dysfunction in zebrafish with potential involvement of O-GlcNAc cycling regulation.

Repeated sublethal hypoxia exposure induces brain inflammation and affects the initiation and progression of cognitive dysfunction. Experiments from the current study showed that hypoxic exposure downregulates PKA/CREB signaling, which is restored by forskolin (FSK), an adenylate cyclase activator, in both Neuro2a (N2a) cells and zebrafish brain. FSK significantly protected N2a cells from hypoxia-induced cell death and neurite shrinkage. Intraperitoneal administration of FSK for 5 days on zebrafish additionally led to significant recovery from hypoxia-induced social interaction impairment and learning and memory (L/M) deficit. FSK suppressed hypoxia-induced neuroinflammation, as indicated by the observed decrease in NF-κB activation and GFAP expression. We further investigated the potential effect of FSK on O-GlcNAcylation changes induced by hypoxia. Intriguingly FSK induced marked upregulation of the protein level of O-GlcNAc transferase catalyzing addition of the GlcNAc group to target proteins, accompanied by elevated O-GlcNAcylation of nucleocytoplasmic proteins. The hypoxia-induced O-GlcNAcylation decrease in the brain of zebrafish was considerably restored following FSK treatment. Based on the collective results, we propose that FSK rescues hypoxia-induced cognitive dysfunction, potentially through regulation of HBP/O-GlcNAc cycling.

Protective effect of increased O-GlcNAc cycling against 6-OHDA induced Parkinson's disease pathology.

This study aimed to elucidate the role of O-GlcNAc cycling in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD)-like neurodegeneration and the underlying mechanisms. We observed dose-dependent downregulation of O-GlcNAcylation, accompanied by an increase in O-GlcNAcase following 6-OHDA treatment in both mouse brain and Neuro2a cells. Interestingly, elevating O-GlcNAcylation through glucosamine (GlcN) injection provided protection against PD pathogenesis induced by 6-OHDA. At the behavioral level, GlcN mitigated motor deficits induced by 6-OHDA, as determined using the pole, cylinder, and apomorphine rotation tests. Furthermore, GlcN attenuated 6-OHDA-induced neuroinflammation and mitochondrial dysfunction. Notably, augmented O-GlcNAcylation, achieved through O-GlcNAc transferase (OGT) overexpression in mouse brain, conferred protection against 6-OHDA-induced PD pathology, encompassing neuronal cell death, motor deficits, neuroinflammation, and mitochondrial dysfunction. These collective findings suggest that O-GlcNAcylation plays a crucial role in the normal functioning of dopamine neurons. Moreover, enhancing O-GlcNAcylation through genetic and pharmacological means could effectively ameliorate neurodegeneration and motor impairment in an animal model of PD. These results propose a potential strategy for safeguarding against the deterioration of dopamine neurons implicated in PD pathogenesis.

O-GlcNAc transferase inhibits LPS-mediated expression of inducible nitric oxide synthase through an increased interaction with mSin3A in RAW264.7 cells.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of a single ß-N-GlcNAc unit to target proteins, has been shown to act as a transcriptional regulator. In the current study, we discovered that OGT exerted inhibitory effects on the LPS-driven activation of NF-κB and inducible nitric oxide synthase (iNOS). In response to LPS, OGT exhibited an increased interaction with the transcriptional corepressor mammalian Sin3A (mSin3A). Furthermore, mSin3A, histone deacetylase (HDAC)1, and HDAC2 displayed increased binding to the iNOS promoter in response to LPS. Treatment with GlcN, in contrast, inhibits LPS-induced inflammation and decreased LPS-mediated recruitment of OGT, mSin3A, and HDACs. LPS treatment also resulted in the hypo-O-GlcNAcylation of mSin3A, which was reversed by GlcN. When the effect of the HDAC inhibitor trichostatin A (TSA) on LPS- and/or GlcN-mediated iNOS protein/mRNA induction was investigated, the results revealed that TSA dose dependently enhanced iNOS expression in response to LPS and/or GlcN. In addition, histone acetyltransferases, p300, and cAMP response element-binding protein-binding protein (CBP) enhanced LPS- and/or GlcN-induced iNOS protein expression. These results collectively suggest that OGT inhibits LPS-driven NF-κB activation and subsequent iNOS transcription by modulating histone acetylation either directly or indirectly.

Melanocortin 1 receptor regulates melanoma cell migration by controlling syndecan-2 expression.

The melanocortin 1 receptor (MC1R), a key regulator of melanogenesis, is known to control inflammation, acting in concert with the MC1R ligand α-melanocyte-stimulating hormone. Although cell migration is a key event in inflammation, few studies have addressed the function of MC1R in this context. Using highly motile melanoma cells, we found that the expression level of MC1R was associated with the extent of migration of mouse melanoma cells, suggesting that MC1R plays a functional role in controlling this migration. Overexpression of MC1R enhanced melanoma cell migration, whereas the opposite was true when MC1R levels were knocked down using small inhibitory RNAs. Interestingly, MC1R expression enhanced the synthesis of syndecan-2, a cell surface heparan sulfate proteoglycan known to be involved in melanoma cell migration. Knockdown of syndecan-2 expression decreased MC1R-mediated cell migration. Further, MC1R inhibited the activation of p38 MAPK, subsequently enhancing expression of sydnecan-2, in parallel with an increase in the extent of cell migration. Consistently, activation of p38 by H(2)O(2) inhibited syndecan-2 expression and cell migration, whereas inhibition of p38 activation enhanced syndecan-2 expression and cell migration. Finally, we found that α-melanocyte-stimulating hormone inhibited MC1R-mediated cell migration via activation of p38 and inhibition of syndecan-2 expression. Together, the data strongly suggest that MC1R regulates melanoma cell migration via inhibition of syndecan-2 expression.

Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

Expression of carbonic anhydrase IX (CA9) was shown to be strongly involved in high incidences of metastasis and poor prognosis in various human tumors. In this study, we investigated the possible role for CA9 in tumor metastases in vitro, using a gene transfection tool in the human cervical carcinoma cell line C33A. Gene expression profiling of CA9-transfected cells (C33A/CA9) and vector-transfected cells (C33A/Mock) was investigated by DNA microarray. The biological functions of differentially expressed genes between the C33A/CA9 and C33A/Mock cells included cell growth, regulation of cell-cell and cell-extracellular matrix adhesion and cytoskeletal organization. Immunofluorescent stain and Matrigel culture showed cytoskeletal remodeling, disassembled focal adhesion, weakened cell-cell adhesion and increased motility in C33A/CA9 cells. These invasive and metastatic phenotypes were associated with Rho-GTPase-related epithelial-mesenchymal transition. Inhibition of the Rho/Rho kinase pathway by a ROCK inhibitor (Y27632) and si-Rho (short interference RNA against RhoA) showed that Rho-GTPase signaling was involved in cellular morphologic and migratory changes. The effect of CA9 on Rho-GTPase signaling was also confirmed by silencing CA9 expression. Our results suggest that CA9 overexpression induces weakening of cell adhesions and augmented cell motility by aberrant Rho-GTPase signal transduction. Our study shows an underlying mechanism of CA9-related enhanced metastatic potential of tumor cells.

Glucosamine inhibits lipopolysaccharide-stimulated inducible nitric oxide synthase induction by inhibiting expression of NF-kappaB/Rel proteins at the mRNA and protein levels.

Expression of inducible nitric oxide synthase (iNOS) protein by lipopolysaccharide (LPS) in BV2 microglia cells increased in a biphasic manner. Glucosamine (GlcN) selectively suppressed the late- but not early-stage iNOS response to LPS. Prolonged induction of iNOS expression by LPS was inhibited by cycloheximide, suggesting that de novo protein synthesis was required. Late-phase activation of nuclear factor-kappaB (NF-κB) activity required for sustained iNOS induction. Nuclear translocation and DNA binding of NF-κB, and Rel proteins expressions were inhibited by GlcN at later time points but not upon immediate early-stage activation by LPS. We show that GlcN selectively inhibits sustained iNOS induction by inhibiting Rel protein expression at both the mRNA and protein levels; such expression is required for prolonged iNOS induction by LPS. Our results provide mechanistic evidence that GlcN regulates inflammation, represented by iNOS. The implication of these results is that GlcN may be a potent transcriptional regulator of iNOS and other genes involved in the general inflammation process.

Synergistic Anti-Inflammatory Effects of Ethanol Extracts from Chrysanthemum zawadskii Flower and Cudrania tricuspidata Fruit Occur via Inhibition of the NF-κB Signaling Pathway.

Chrysanthemum zawadskii (CZ) and Cudrania tricuspidata (CT) are both traditional Korea herbal medicines, which is widely used to treat fever, cough, gastritis, and women's diseases that may be linked to inflammatory response. Although it has been used to treat diseases related to inflammation, there has been no case of the synergistic anti-inflammatory properties of both extracts. Our data revealed that ethanol extracts of dried whole CZ exhibited free radical-scavenging capacity in vitro, reduced LPS-induced intracellular reactive oxygen species, and decreased the LPS-induced upregulations of the mRNAs encoding iNOS, COX-2, and IL-6 in RAW 264.7 cells, without significant cytotoxicity. This anti-inflammatory effect was most evident from flower extracts: ethanol extracts from flowers significantly reduced the LPS-induced upregulations of iNOS and COX-2 at a concentration of 100 µg/ml. An ethanol extract of the fruit from CT also exerted a radical scavenging capacity and suppressed LPS-induced proinflammatory gene expression: 5.5 µg/ml of the ethanol extract significantly reduced the ability of LPS to induce the mRNA expression levels of iNOS and IL-6 without apparent cytotoxicity. Furthermore, as little as 1.0 µg/ml of the combined ethanol extracts of CZ flower and CT fruit reduced the LPS-induced changes monitored herein, decreasing the upregulations of iNOS and IL-6, and decreasing the nuclear localization of NF-κB p65. These results suggest that the observed synergistic anti-inflammatory effects may be mediated via inhibition of NF-κB signaling. Taken together, these data suggest that ethanol extracts from CZ flowers and CT fruits have synergistic anti-inflammatory effects and that a combination of the two extracts could prove useful for the treatment of inflammation-related diseases.

Keratinocyte-derived laminin-332 promotes adhesion and migration in melanocytes and melanoma.

Melanocytes are highly motile cells that play an integral role in basic skin physiological processes such as wound healing and proper skin pigmentation. It has been postulated that surrounding keratinocytes contribute to melanocyte migration, but underlying mechanisms remain rather vague so far. In this study, we set out to analyze the specific potential contribution of keratinocyte components to melanocytes and melanoma cell migration-related processes. Our studies revealed that A375 human melanoma cell attachment, spreading, and migration are interestingly better supported by HaCaT keratinocyte extracellular matrix (ECM) than by self-derived A375 ECM. Moreover, HaCaT ECM caused increased integrin α6 expression, adhesion-mediated focal adhesion kinase phosphorylation, and focal adhesion formations. Similar effects were confirmed in human melanocytes. Furthermore, we found that keratinocyte-derived soluble factors did not appear to significantly contribute to these processes. Specific extrinsic factors that promoted melanoma migration were attributed to keratinocyte-derived laminin-332, whereas alternative ECM component such as laminin-111 and fibronectin functions appeared to have insignificant contributions. Taken together, these studies implicate extrinsic laminin-332 in promoting the high mobility property and perhaps invasiveness inherently characteristic of, and that are the menace of, melanocytes and melanomas, respectively.