Pontin Functions as A Transcriptional Co-activator for Retinoic Acid-induced HOX Gene Expression.

Pontin is a AAA+ ATPase protein that has functions in various biological contexts including gene transcription regulation, chromatin remodeling, DNA damage sensing and repair, as well as assembly of protein and ribonucleoprotein complexes. Pontin is known to regulate the transcription of several important signaling pathways, including Wnt signaling. However, its role in early embryonic signaling regulation remains unclear. Retinoic acid (RA) signaling plays a central role in vertebrate development. Using an in vivo biotin tagging technology, we mapped the genome-wide binding pattern of Pontin before and after RA-induced differentiation in the pluripotent embryo carcinoma cell line NTERA-2. Biotin ChIP-seq revealed significant changes in genome-wide Pontin binding sites upon RA stimulation. We also identified a substantial amount of overlapping binding peaks between Pontin and RARα, especially on all of the HOX gene loci (A-D clusters). Pontin knockdown experiments showed that its chromatin binding at the HOX gene clusters is required for RA-induced HOX gene expression. Furthermore, we performed Global Run-On sequencing (GRO-seq) to map de novo transcripts genome-wide and found that Pontin knockdown significantly diminished nascent HOX gene transcripts, indicating that Pontin regulates HOX gene expression at the transcriptional level. Finally, proteomic analysis demonstrated that Pontin associates with chromatin organization/remodeling complexes and various other functional complexes. Altogether, we have demonstrated that Pontin is a critical transcriptional co-activator for RA-induced HOX gene activation.

Homoarginine ameliorates diabetic nephropathy independent of nitric oxide synthase-3.

Recently we showed that homoarginine supplementation confers kidney protection in diabetic mouse models. In this study we tested whether the protective effect of homoarginine is nitric oxide synthase-3 (NOS3)-independent in diabetic nephropathy (DN). Experiments were conducted in NOS3 deficient (NOS3-/- ) mice and their wild type littermate using multiple low doses of vehicle or streptozotocin and treated with homoarginine via drinking water for 24 weeks. Homoarginine supplementation for 24 weeks in diabetic NOS3-/- mice significantly attenuated albuminuria, increased blood urea nitrogen, histopathological changes and kidney fibrosis, kidney fibrotic markers, and kidney macrophage recruitment compared with vehicle-treated diabetic NOS3-/- mice. Furthermore, homoarginine supplementation restored kidney mitochondrial function following diabetes. Importantly, there were no significant changes in kidney NOS1 or NOS2 mRNA expression between all groups. In addition, homoarginine supplementation improved cardiac function and reduced cardiac fibrosis following diabetes. These data demonstrate that the protective effect of homoarginine is independent of NOS3, which will ultimately change our understanding of the mechanism(s) by which homoarginine induce renal and cardiac protection in DN. Homoarginine protective effect in DN could be mediated via improving mitochondrial function.

Metal chelation reduces skin epithelial inflammation and rescues epithelial cells from toxicity due to thermal injury in a rat model.

BACKGROUND: One of the most pervasive complications of burn injury is wound progression, characterized by continuous tissue destruction in untreated wounds, which leads to wound infection, inflammation, oxidative stress and excessive scar formation. We determined whether additional tissue destruction could be attenuated with Livionex formulation (LF) lotion, which contains a metal-chelating agent and reduces inflammation in burn wounds. METHODS: We subjected male Sprague Dawley rats to a 2% total body surface area (TBSA) burn using a brass comb model and topically applied LF lotion (containing ethylenediaminetetraacetic acid and methyl sulfonyl methane) to the affected area every 8 hours over 3 days. Inflammatory cytokine levels, cell apoptosis and wound healing were compared in LF lotion-treated and untreated rats. Statistical analysis was performed using a one-way analysis of variance in conjunction with Tukey's post-hoc test. RESULTS: Serum inflammatory cytokines were not detectable after 3 days, suggesting that small burn wounds induce only an immediate, localized inflammatory response. Microscopy revealed that LF lotion improved burn site pathology. Deoxynucleotidyl transferase biotin-d-UTP nick-end labeling staining showed reduced cell death in the LF-treated samples. LF lotion prevented the spread of tissue damage, as seen by increased amounts of Ki-67-positive nuclei in the adjacent epidermis and hair follicles. Tumor necrosis factor-alpha, interleukin-6 and inducible nitric oxide synthase levels in LF-treated skin sections from burned rats were comparable to the levels observed in unburned control sections, indicating that LF lotion reduces inflammation in and around the burn site. CONCLUSIONS: These results establish LF lotion as a therapeutic agent for reducing inflammatory stress, cell death and tissue destruction when applied immediately after a burn injury. Further studies of LF lotion on large TBSA burns will determine its efficacy as an emergency treatment for reducing long-term morbidity and scarring.

Selective inhibition of arginase-2 in endothelial cells but not proximal tubules reduces renal fibrosis.

Fibrosis is the final common pathway in the pathophysiology of most forms of chronic kidney disease (CKD). As treatment of renal fibrosis still remains largely supportive, a refined understanding of the cellular and molecular mechanisms of kidney fibrosis and the development of novel compounds are urgently needed. Whether arginases play a role in the development of fibrosis in CKD is unclear. We hypothesized that endothelial arginase-2 (Arg2) promotes the development of kidney fibrosis induced by unilateral ureteral obstruction (UUO). Arg2 expression and arginase activity significantly increased following renal fibrosis. Pharmacologic blockade or genetic deficiency of Arg2 conferred kidney protection following renal fibrosis, as reflected by a reduction in kidney interstitial fibrosis and fibrotic markers. Selective deletion of Arg2 in endothelial cells (Tie2Cre/Arg2fl/fl) reduced the level of fibrosis after UUO. In contrast, selective deletion of Arg2 specifically in proximal tubular cells (Ggt1Cre/Arg2fl/fl) failed to reduce renal fibrosis after UUO. Furthermore, arginase inhibition restored kidney nitric oxide (NO) levels, oxidative stress, and mitochondrial function following UUO. These findings indicate that endothelial Arg2 plays a major role in renal fibrosis via its action on NO and mitochondrial function. Blocking Arg2 activity or expression could be a novel therapeutic approach for prevention of CKD.

Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.

l-Homoarginine supplementation prevents diabetic kidney damage.

l-homoarginine is an endogenous, non-proteinogenic amino acid that has emerged as a new player in health and disease. Specifically, low l-homoarginine levels are associated with cardiovascular diseases, stroke, and reduced kidney function. However, the role of l-homoarginine in the pathogenesis of diabetic nephropathy (DN) is not known. Experiments were conducted in 6-week-old Ins2Akita mice supplemented with l-homoarginine via drinking water or mini osmotic pump for 12 weeks. Both plasma and kidney l-homoarginine levels were significantly reduced in diabetic mice compared to nondiabetic controls. Untreated Ins2Akita mice showed significant increases in urinary albumin excretion, histological changes, glomerular macrophage recruitment, the inflammatory cytokine KC-GRO/CXCL1, and urinary thiobarbituric acid reactive substances (TBARS) excretion as an indicator of oxidative stress, along with a significant reduction in kidney nitrate + nitrite levels compared to control mice at 18 weeks of age. In contrast, l-homoarginine supplementation for 12 weeks in Ins2Akita mice, via either drinking water or mini osmotic pump, significantly reduced albuminuria, renal histological changes, glomerular macrophage recruitment, KC-GRO/CXCL1 levels, urinary TBARS excretion, and largely restored kidney nitrate + nitrite levels. These data demonstrate that l-homoarginine supplementation attenuates specific features of DN in mice and could be a potential new therapeutic tool for treating diabetic patients.

Mechanisms by which hydrogen sulfide attenuates muscle function following ischemia-reperfusion injury: effects on Akt signaling, mitochondrial function, and apoptosis.

Ischemia-reperfusion injury is caused by a period of ischemia followed by massive blood flow into a tissue that had experienced restricted blood flow. The severity of the injury is dependent on the time the tissue was restricted from blood flow, becoming more severe after longer ischemia times. This can lead to many complications such as tissue necrosis, cellular apoptosis, inflammation, metabolic and mitochondrial dysfunction, and even organ failure. One of the emerging therapies to combat ischemic reperfusion injury complications is hydrogen sulfide, which is a gasotransmitter that diffuses across cell membranes to exert effects on various signaling pathways regulating cell survival such as Akt, mitochondrial activity, and apoptosis. Although commonly thought of as a toxic gas, low concentrations of hydrogen sulfide have been shown to be beneficial in promoting tissue survival post-ischemia, and modulate a wide variety of cellular responses. This review will detail the mechanisms of hydrogen sulfide in affecting the Akt signaling pathway, mitochondrial function, and apoptosis, particularly in regards to ischemic reperfusion injury in muscle tissue. It will conclude with potential clinical applications of hydrogen sulfide, combinations with other therapies, and perspectives for future studies.

Buprenorphine-Sustained Release Alters Hemodynamic Parameters in a Rat Burn Model.

BACKGROUND: It has been previously shown that anesthesia and analgesia can affect outcomes in the rat burn model and that buprenorphine alleviated pain without drastically altering the outcomes of interest. Recently, the use of a sustained release (SR) formulation of buprenorphine has been promoted over conventional buprenorphine. In this study, we assessed whether buprenorphine-SR altered hemodynamic parameters in our rat model of severe burn injury. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were randomized to receive either conventional buprenorphine (0.05 mg/kg) or buprenorphine-SR (1 mg/kg). Buprenorphine-SR was administered 24 h before the experiment. Buprenorphine was administered on the day of experiment. These groups were further randomized to control or scald burn (60% of total body surface area). Systolic and diastolic blood pressure (SBP, DBP) and heart rate (HR) were measured using a noninvasive blood pressure system before receiving analgesia and after 72 h. RESULTS: As expected, HR was significantly higher after burn injury regardless of analgesic (P <0.0001). Both SBP and DBP were significantly decreased in burned animals receiving conventional buprenorphine (P < 0.0001), but neither was altered in the buprenorphine-SR-treated burned animals. However, SBP, DBP, and HR were significantly increased after 72 h in control animals receiving buprenorphine-SR (P < 0.0001). CONCLUSIONS: These data indicate that buprenorphine-SR alters the hemodynamic response to injury and may not be an appropriate choice for a model of severe burn injury. If this analgesic is used, investigators must cautiously form conclusions, especially in experimental conditions that would be expected to alter cardiac hemodynamics.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice.

There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.

Characterization of Adipose-Derived Stem Cells Following Burn Injury.

Severe burns induce a prolonged inflammatory response in subcutaneous adipose tissue that modulates signaling in adipose-derived stem cells (ASCs), which hold potential for healing burn wounds or generating skin substitutes. Using a 60% rat scald burn model, we conducted a series of experiments to determine which cells isolated from the adipose tissue produced inflammatory mediators and how these changes affect ASC fate and function. The stromal vascular fraction (SVF), adipocytes, and ASCs were isolated from adipose tissue at varying times up to 4 weeks postburn and from non-injured controls. Endpoints included inflammatory marker expression, expression of ASC-specific cell-surface markers, DNA damage, differentiation potential, and proliferation. Inflammatory marker expression was induced in adipocytes and the SVF at 24 and 48 h postburn; expression of inflammatory marker mRNA transcripts and protein returned to normal in the SVF isolated 1 week postburn. In enriched ASCs, burns did not alter cell-surface expression of stem cell markers, markers of inflammation, differentiation potential, or proliferative ability. These results suggest that adipocytes and the SVF produce large quantities of inflammatory mediators, but that ASCs do not, after burns and that ASCs are unaffected by burn injury or culturing procedures.. They also suggest that cells isolated over 48 h after injury are best for cell culture or tissue engineering purposes.

Absence of CCR2 results in an inflammaging environment in young mice with age-independent impairments in muscle regeneration.

Skeletal muscle regeneration requires coordination between dynamic cellular populations and tissue microenvironments. Macrophages, recruited via CCR2, are essential for regeneration; however, the contribution of macrophages and the role of CCR2 on nonhematopoietic cells has not been defined. In addition, aging and sex interactions in regeneration and sarcopenia are unclear. Muscle regeneration was measured in young (3-6 mo), middle (11-15 mo), old (24-32 mo) male and female CCR2-/- mice. Whereas age-related muscle atrophy/sarcopenia was present, regenerated myofiber cross-sectional area (CSA) in CCR2-/- mice was comparably impaired across all ages and sexes, with increased adipocyte area compared with wild-type (WT) mice. CCR2-/- mice myofibers achieved approximately one third of baseline CSA even 84 d after injury. Regenerated CSA and clearance of necrotic tissue were dependent on bone marrow-derived cellular expression of CCR2. Myogenic progenitor cells isolated from WT and CCR2-/- mice exhibited comparable proliferation and differentiation capacity. The most striking cellular anomaly in injured muscle of CCR2-/- mice was markedly decreased macrophages, with a predominance of Ly6C- anti-inflammatory monocytes/macrophages. Ablation of proinflammatory TLR signaling did not affect muscle regeneration or resolution of necrosis. Of interest, many proinflammatory, proangiogenic, and chemotactic cytokines were markedly elevated in injured muscle of CCR2-/- relative to WT mice despite impairments in macrophage recruitment. Collectively, these results suggest that CCR2 on bone marrow-derived cells, likely macrophages, were essential to muscle regeneration independent of TLR signaling, aging, and sex. Decreased proinflammatory monocytes/macrophages actually promoted a proinflammatory microenvironment, which suggests that inflammaging was present in young CCR2-/- mice.

Effects of the nephrilin peptide on post-burn glycemic control, renal function, fat and lean body mass, and wound healing.

The mechanisms underlying the effects of severe burn trauma are not well understood. We previously demonstrated the ability of nephrilin peptide (an iron-binding peptide believed to enter cells through iron-uptake pathways) to suppress aspects of the neuroinflammatory response in a rat scald model, as well as sepsis mortality in a mouse model. This study explores the effect of nephrilin on other clinically relevant outcomes in the rat scald model. In a rat scald model, animals were treated with nephrilin either in week 1 or week 2 post-burn. Measurements were made of serum glucose and creatinine as well as wound area by planimetry and body composition by DEXA. Given the potential role of iron, results were analyzed both for the entire cohort of animals and for the normoferremic (>100 ug/dL serum iron) subset of animals. Nephrilin improved body composition, wound healing, kidney function, and glycemic control. The first two effects were significant in normoferremic but not in hypoferremic animals suggesting an effect of iron status on burn injury outcomes. Nephrilin treatment modulates a number of relevant variables in the rat scald model.

Heparanase mediates a novel mechanism in lapatinib-resistant brain metastatic breast cancer.

Heparanase (HPSE) is the dominant mammalian endoglycosidase and important tumorigenic, angiogenic, and pro-metastatic molecule. Highest levels of HPSE activity have been consistently detected in cells possessing highest propensities to colonize the brain, emphasizing the therapeutic potential for targeting HPSE in brain metastatic breast cancer (BMBC). Lapatinib (Tykerb) is a small-molecule and dual inhibitor of human epidermal growth factor receptor1 and 2 (EGFR and HER2, respectively) which are both high-risk predictors of BMBC. It was approved by the US Food and Drug Administration for treatment of patients with advanced or metastatic breast cancer. However, its role is limited in BMBC whose response rates to lapatinib are significantly lower than those for extracranial metastasis. Because HPSE can affect EGFR phosphorylation, we examined Roneparstat, a non-anticoagulant heparin with potent anti-HPSE activity, to inhibit EGFR signaling pathways and BMBC onset using lapatinib-resistant clones generated from HER2-transfected, EGFR-expressing MDA-MB-231BR cells. Cell growth, EGFR pathways, and HPSE targets were assessed among selected clones in the absence or presence of Roneparstat and/or lapatinib. Roneparstat overcame lapatinib resistance by inhibiting pathways associated with EGFR tyrosine residues that are not targeted by lapatinib. Roneparstat inhibited the growth and BMBC abilities of lapatinib-resistant clones. A molecular mechanism was identified by which HPSE mediates an alternative survival pathway in lapatinib-resistant clones and is modulated by Roneparstat. These results demonstrate that the inhibition of HPSE-mediated signaling plays important roles in lapatinib resistance, and provide mechanistic insights to validate the use of Roneparstat for novel BMBC therapeutic strategies.

iMatrixSpray: a free and open source sample preparation device for mass spectrometric imaging.

A device was built for matrix deposition in mass spectrometric imaging. This spray-type instrument requires no user interaction other than providing the spray solution and selecting the pre-defined or custom-built method. Robustness was achieved by utilizing a delta-robotics design in combination with a simple liquid system. All the information describing the systems is provided as open source and hardware and the design is therefore suitable for wide distribution and adaption by the scientific community.

The identification and characterization of breast cancer CTCs competent for brain metastasis.

Brain metastatic breast cancer (BMBC) is uniformly fatal and increasing in frequency. Despite its devastating outcome, mechanisms causing BMBC remain largely unknown. The mechanisms that implicate circulating tumor cells (CTCs) in metastatic disease, notably in BMBC, remain elusive. We characterize CTCs isolated from peripheral blood mononuclear cells of patients with breast cancer and also develop CTC lines from three of these patients. In epithelial cell adhesion molecule (EpCAM)-negative CTCs, we identified a potential signature of brain metastasis comprising "brain metastasis selected markers (BMSMs)" HER2+ / EGFR+ / HPSE+ / Notch1+. These CTCs, which are not captured by the CellSearch platform because of their EpCAM negativity, were analyzed for cell invasiveness and metastatic competency in vivo. CTC lines expressing the BMSM signature were highly invasive and capable of generating brain and lung metastases when xenografted in nude mice. Notably, increased brain metastatic capabilities, frequency, and quantitation were detected in EpCAM- CTCs overexpressing the BMSM signature. The presence of proteins of the BMSM CTC signature was also detected in the metastatic lesions of animals. Collectively, we provide evidence of isolation, characterization, and long-term culture of human breast cancer CTCs, leading to the description of a BMSM protein signature that is suggestive of CTC metastatic competency to the brain.

Heparanase-induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells.

Heparanase is the only mammalian endoglycosidase which has been widely implicated in cancer because of its capability to degrade heparan sulfate chains of heparan sulfate proteoglycans (HSPG). Specifically, the cell surface HSPG syndecan-1 and -4 (SDC1 and SDC4) are modulators of growth factor action, and SDC4 is implicated in cell adhesion as a key member of focal adhesion complexes. We hypothesized that extracellular heparanase modulates brain metastatic breast cancer (BMBC) cell invasiveness by affecting cytoskeletal dynamics, SDC4 carboxy-terminal-associated proteins, and downstream targets. We used two independently derived human BMBC cell systems (MB-231BR and MB-231BR3), which possess distinct cellular morphologies and properties. Highly aggressive spindle-shaped 231BR3 cells changed to a round cell morphology associated with expression of the small GTPase guanine nucleotide exchange factor-H1 (GEF-H1). We showed that GEF-H1 is a new component of the SDC4 signaling complex in BMBC cells. Treatment with heparanase resulted in regulation of the SDC4/protein kinase C α axis while maintaining a constitutive GEF-H1 level. Third, GEF-H1 knockdown followed by cell exposure to heparanase caused a significant regulation of activities of Rac1 and RhoA, which are GEF-H1 targets and fundamental effectors in cell plasticity control. Fourth, L-heparanase augmented expression of ß1 integrin in BMBC cells and of vascular cell adhesion molecule 1 (VCAM1; the major ß1 integrin receptor) in human brain microvascular endothelial cells. Finally, using a newly developed blood-brain barrier in vitro model, we show that BMBC cell transmigration was significantly reduced in GEF-H1 knockdown cells. These findings implicate heparanase in mechanisms of cytoskeletal dynamics and in the cross-talk between tumor cells and vascular brain endothelium. They are of relevance because they elucidate molecular events in the initial steps leading to BMBC onset and capturing distinct roles of latent and active heparanase in the brain microenvironment.

Heparanase Modulates Shh and Wnt3a Signaling in Human Medulloblastoma Cells.

The pathogenesis of medulloblastoma (MB), the most common and aggressive brain tumor in children, is poorly understood. MB tumors respond to factors secreted by cerebellar Purkinje neurons such as Sonic hedgehog (Shh) and Wnt3a. Understanding the modulation of Shh/Wnt signaling is critical to developing new MB treatments. Shh and Wnt3a induce MB cell proliferation, and bind heparan sulfate glycosaminoglycan chains (HS-GAG). HS-GAG are components of syndecans: cell surface HS proteoglycans (HSPG) which act as co-receptors for extracellular matrix based ligands, and are targets of heparanase (HPSE). We hypothesized that extracellular HPSE activity can modulate MB intracellular signaling of Shh/Wnt3a, involving syndecans 1/4 carboxy terminal-associated proteins and downstream targets. We compared the regulation of Shh/Wnt3a signaling subsequent to treatment with exogenous human active HPSE in MB lines possessing increased invasive abilities. We identified GEF-H1, a small GTPase guanine nucleotide exchange factor, as a new component of a syndecan signaling complex. Secondly, we demonstrated that HPSE modulated Shh/Wnt3 dependent expression and intracellular distribution of GEF-H1, ß-catenin, and N-Myc. Thirdly, HPSE modulated Shh/Wnt3a - dependent gene expression of HSPG and Gli transcription factors. Fourthly, pretreatment with HPSE, alone or prior to Shh/Wnt3a exposure, altered small GTPase (Rac1/RhoA) activities differentially, and promoted RhoA activation. Finally, the differential regulation of Rac1/RhoA activities by HPSE affected MB cell proliferation and invasion. Our results indicate that the HPSE/HSPG axis is implicated in critical MB cell signaling pathways with potential relevance for MB treatment.

Modulation of GEF-H1 induced signaling by heparanase in brain metastatic melanoma cells.

Mechanisms of brain metastatic melanoma (BMM) remain largely unknown. Understanding the modulation of signaling pathways that alter BMM cell invasion and metastasis is critical to develop new therapies for BMM. Heparanase has been widely implicated in cancer and is the dominant mammalian endoglycosidase which degrades heparan sulfate chains of proteoglycans (HSPG) including syndecans (SDCs). Recent findings also indicate that heparanase possesses non-enzymatic functions in its latent form. We hypothesized that extracellular heparanase modulates BMM cell signaling by involving SDC1/4 carboxy terminal-associated proteins and downstream targets. We digested BMM cell surface HS with human recombinant active or latent heparanase to delineate their effects on cytoskeletal dynamics and cell invasiveness. We identified the small GTPase guanine nucleotide exchange factor-H1 (GEF-H1) as a new component of a SDC signaling complex that is differentially expressed in BMM cells compared to corresponding non-metastatic counterparts. Second, knockdown of GEF-H1, SDC1, or SDC4 decreased BMM cell invasiveness and GEF-H1 modulated small GTPase activity of Rac1 and RhoA in conjunction with heparanase treatment. Third, both active and latent forms of heparanase affected Rac1 and RhoA activity; notably increasing RhoA activity. Both forms of heparanase were found to mediate the expression and subcellular localization of GEF-H1, and treatment of BMM with latent heparanase modulated SDC1/4 gene expression. Finally, treatment with exogenous heparanase downregulated BMM cell invasion. These studies indicate the relevance of heparanase signaling pathways in BMM progression, and provide insights into the molecular mechanisms regulating HSPG signaling in response to exogenous heparanase.

A disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates adiponectin multimerization.

Impairments in adiponectin multimerization lead to defects in adiponectin secretion and function and are associated with diabetes, yet the underlying mechanisms remain largely unknown. We have identified an adiponectin-interacting protein, previously named GST-kappa, by yeast 2-hybrid screening. The adiponectin-interacting protein contains 2 thioredoxin domains and has very little sequence similarity to other GST isoforms. However, this protein shares high sequence and secondary structure homology to bacterial disulfide-bond A oxidoreductase (DsbA) and is thus renamed DsbA-like protein (DsbA-L). DsbA-L is highly expressed in adipose tissue, and its expression level is negatively correlated with obesity in mice and humans. DsbA-L expression in 3T3-L1 adipocytes is stimulated by the insulin sensitizer rosiglitazone and inhibited by the inflammatory cytokine TNFalpha. Overexpression of DsbA-L promoted adiponectin multimerization while suppressing DsbA-L expression by RNAi markedly and selectively reduced adiponectin levels and secretion in 3T3-L1 adipocytes. Our results identify DsbA-L as a key regulator for adiponectin biosynthesis and uncover a potential new target for developing therapeutic drugs for the treatment of insulin resistance and its associated metabolic disorders.

Adiponectin sensitizes insulin signaling by reducing p70 S6 kinase-mediated serine phosphorylation of IRS-1.

Adiponectin functions as an insulin sensitizer, and yet the underlying molecular mechanism(s) remains largely unknown. We found that treating C2C12 myotubes with adiponectin or rapamycin enhanced the ability of insulin to stimulate IRS-1 tyrosine phosphorylation and Akt phosphorylation, concurrently with reduced p70 S6 kinase phosphorylation at Thr389 as well as IRS-1 phosphorylation at Ser302 and Ser636/639. Overexpression of dominant-negative AMP kinase (AMPK), but not dominant-negative p38 MAPK, reduced the insulin-sensitizing effect of adiponectin. Rapamycin, but not adiponectin, enhanced insulin-stimulated Akt phosphorylation in HeLa cells, which lack LKB1, and exogenous expression of LKB1 in HeLa cells rescued the insulin-sensitizing effect of adiponectin. Finally, overexpression of wild-type Rheb (Ras homology-enriched in brain) or the TSC2 mutant lacking the AMPK phosphorylation site (TSC2S1345A) inhibited the insulin-sensitizing effect of adiponectin in C2C12 cells. These results indicate that activation of the LKB1/AMPK/TSC1/2 pathway alleviates the p70 S6 kinase-mediated negative regulation of insulin signaling, providing a mechanism by which adiponectin increases insulin sensitivity in cells.