A novel biological function of soluble biglycan: Induction of erythropoietin production and polycythemia

Secondary polycythemia, a disease characterized by a selective increase in circulating mature erythrocytes, is caused by enhanced erythropoietin (Epo) concentrations triggered by hypoxia-inducible factor-2α (HIF-2α). While mechanisms of hypoxia-dependent stabilization of HIF-2α protein are well established, data regarding oxygen-independent regulation of HIF-2α are sparse. In this study, we generated a novel transgenic mouse model, in which biglycan was constitutively overexpressed and secreted by hepatocytes (BGN ^Tg), thereby providing a constant source of biglycan released into the blood stream. We discovered that although the mice were apparently normal, they harbored an increase in mature circulating erythrocytes. In addition to erythrocytosis, the BGN ^Tg mice showed elevated hemoglobin concentrations, hematocrit values and enhanced total iron binding capacity, revealing a clinical picture of polycythemia. In BGN ^Tg mice markedly enhanced Epo mRNA expression was observed in the liver and kidney, while elevated Epo protein levels were found in liver, kidney and blood. Mechanistically, we showed that the transgenic animals had an abundance of HIF-2α protein in the liver and kidney. Finally, by transiently overexpressing circulating biglycan in mice deficient in various Toll-like receptors (TLRs), we determined that this novel function of biglycan to promote Epo synthesis was specifically mediated by a selective interaction with TLR2. Thus, we discovered a novel biological pathway of soluble biglycan inducing HIF-2α protein stabilization and Epo production presumably in an oxygen-independent manner, ultimately giving rise to secondary polycythemia.     

Neoplastic modulation of extracellular matrix. Colon carcinoma cells release polypeptides that alter proteoglycan metabolism in colon fibroblasts

Despite the growing evidence implicating proteoglycans in the control of cell proliferation and differentiation, little is known about the factors that control their metabolism in neoplasia or the mechanisms through which these macromolecules may influence neoplastic growth. The primary objective of the present study was to test whether human colon carcinoma cells released soluble mediators capable of stimulating the synthesis of proteoglycans in normal colon fibroblasts in vitro. Serum-free medium conditioned by colon carcinoma cells (TCM) was capable of stimulating several-fold the synthesis and secretion of proteoglycans in normal colon fibroblasts without inducing a mitogenic response. This effect was a true stimulation of proteoglycan biosynthesis since the kinetics of turnover were identical in the presence or absence of TCM. Characterization of the proteoglycans synthesized in the absence of TCM revealed that colon fibroblasts synthesized at least three species of proteoglycans including a heparan sulfate proteoglycan which was associated primarily with the cell layer and two populations of proteoglycans which were predominantly released into the medium and contained chondroitin-dermatan sulfate side chains. When fibroblasts were exposed to TCM, they synthesized and released higher amounts of proteoglycans which had overall similar density, molecular weight, and polydispersity but differed from controls in that they contained significantly higher proportions of chondroitin sulfate side chains. Partial characterization of TCM strongly indicated that the stimulatory activity comprised a family of polypeptides, with molecular weight between 5.4 and 6.0 X 10(5), which were heat stable and acid/alkali labile. Neoplastic modulation of proteoglycan metabolism in normal mesenchymal cells may represent an additional mechanism through which tumor cells can alter their surrounding environment.     

The Dermatan Sulfate Proteoglycan Decorin Modulates ��2��1 Integrin and the Vimentin Intermediate Filament System during Collagen Synthesis

Decorin, a small leucine-rich proteoglycan harboring a dermatan sulfate chain at its N-terminus, is involved in regulating matrix organization and cell signaling. Loss of the dermatan sulfate of decorin leads to an Ehlers-Danlos syndrome characterized by delayed wound healing. Decorin-null (Dcn^−/−) mice display a phenotype similar to that of EDS patients. The fibrillar collagen phenotype of Dcn^−/− mice could be rescued in vitro by decorin but not with decorin lacking the glycosaminoglycan chain. We utilized a 3D cell culture model to investigate the impact of the altered extracellular matrix on Dcn^−/− fibroblasts. Using 2D gel electrophoresis followed by mass spectrometry, we identified vimentin as one of the proteins that was differentially upregulated by the presence of decorin. We discovered that a decorin-deficient matrix leads to abnormal nuclear morphology in the Dcn^−/− fibroblasts. This phenotype could be rescued by the decorin proteoglycan but less efficiently by the decorin protein core. Decorin treatment led to a significant reduction of the α2β1 integrin at day 6 in Dcn^−/− fibroblasts, whereas the protein core had no effect on β1. Interestingly, only the decorin core induced mRNA synthesis, phosphorylation and de novo synthesis of vimentin indicating that the proteoglycan decorin in the extracellular matrix stabilizes the vimentin intermediate filament system. We could support these results in vivo, because the dermis of wild-type mice have more vimentin and less β1 integrin compared to Dcn^−/−. Furthermore, the α2β1 null fibroblasts also showed a reduced amount of vimentin compared to wild-type. These data show for the first time that decorin has an impact on the biology of α2β1 integrin and the vimentin intermediate filament system. Moreover, our findings provide a mechanistic explanation for the reported defects in wound healing associated with the Dcn^−/− phenotype.     

The αvβ6 Integrin Is Transferred Intercellularly via Exosomes

Exosomes, cell-derived vesicles of endosomal origin, are continuously released in the extracellular environment and play a key role in intercellular crosstalk. In this study, we have investigated whether transfer of integrins through exosomes between prostate cancer (PrCa) cells occurs and whether transferred integrins promote cell adhesion and migration. Among others, we have focused on the α_vβ₆ integrin, which is not detectable in normal human prostate but is highly expressed in human primary PrCa as well as murine PrCa in Pten^pc−/− mice. After confirming the fidelity of the exosome preparations by electron microscopy, density gradient, and immunoblotting, we determined that the α_vβ₆ integrin is actively packaged into exosomes isolated from PC3 and RWPE PrCa cell lines. We also demonstrate that α_vβ₆ is efficiently transferred via exosomes from a donor cell to an α_vβ₆-negative recipient cell and localizes to the cell surface. De novo α_vβ₆ expression in an α_vβ₆-negative recipient cell is not a result of a change in mRNA levels but is a consequence of exosome-mediated transfer of this integrin between different PrCa cells. Recipient cells incubated with exosomes containing α_vβ₆ migrate on an α_vβ₆ specific substrate, latency-associated peptide-TGFβ, to a greater extent than cells treated with exosomes in which α_vβ₆ is stably or transiently down-regulated by shRNA or siRNA, respectively. Overall, this study shows that exosomes from PrCa cells may contribute to a horizontal propagation of integrin-associated phenotypes, which would promote cell migration, and consequently, metastasis in a paracrine fashion.     

Scleroderma-like properties of skin from caveolin-1-deficient mice: Implications for new treatment strategies in patients with fibrosis and systemic sclerosis

Caveolin-1 (Cav-1), the principal structural component of caveolae, participates in the pathogenesis of several fibrotic diseases, including systemic sclerosis (SSc). Interestingly, affected skin and lung samples from patients with SSc show reduced levels of Cav-1, as compared to normal skin. In addition, restoration of Cav-1 function in skin fibroblasts from SSc patients reversed their pro-fibrotic phenotype. Here, we further investigated whether Cav-1 mice are a useful preclinical model for studying the pathogenesis of SSc. For this purpose, we performed quantitative transmission electron microscopy, as well as biochemical, biomechanical, and immuno-histochemical analysis, of the skin from Cav-1^-/- null mice. Using these complementary approaches, we now show that skin from Cav-1 null mice exhibits many of the same characteristics as SSc skin from patients. These changes include a decrease in collagen fiber diameter, increased maximum stress (a measure tensile strength) and modulus (a measure of stiffness), as well as mononuclear cell infiltration. Furthermore, an increase in autophagy/mitophagy was observed in the stromal cells of the dermis from Cav-1^-/- mice. These findings suggest that changes in cellular energy metabolism (e.g., a shift towards aerobic glycolysis) in these stromal cells may provide a survival mechanism in this “hostile” or pro-inflammatory microenvironment. Taken together, our results demonstrate that Cav-1^-/- mice are a valuable new pre-clinical model for studying scleroderma. Most importantly, our results suggest that inhibition of autophagy and/or aerobic glycolysis may represent a new promising therapeutic strategy for halting fibrosis in SSc patients. Finally, Cav-1^-/- mice are also a pre-clinical model for a “lethal” tumor microenvironment, possibly explaining the link between fibrosis, tumor progression and cancer metastasis.Key words: caveolin-1, skin, scleroderma, systemic sclerosis, fibrosis, preclinical model, metabolism, matrix, autophagy, mitophagy     

Human obesity is characterized by defective fat storage and enhanced muscle fatty acid oxidation, and trimetazidine gradually counteracts these abnormalities

An impaired ability to store fatty acids (FA) in subcutaneous adipose tissue (SAT) may be implicated in the pathogenesis of obesity-related diseases via overexposure of lean tissues and production of free radicals from FA oxidation (FAO). We studied regional FA metabolism in skeletal muscle and adipose tissue in humans and investigated the long-term effects of the FAO inhibitor trimetazidine on glucose and FA metabolism. Positron emission tomography (PET) and [(11)C]palmitate were used to compare FA metabolism in SAT and skeletal muscle between eight obese and eight nonobese subjects (BMI ≥/< 30 kg/m(2)). A subgroup of nine subjects underwent a 1-mo trimetazidine administration. PET with [(11)C]palmitate and [(18)F]fluorodeoxyglucose, indirect calorimetry, and MRI before and after this period were performed to characterize glucose and FA metabolism, fat masses, skeletal muscle triglyceride, and creatine contents. Obesity was characterized by a 100% elevation in FAO and a defect in the FA esterification rate constant (P < 0.05) in skeletal muscle. FA esterification was reduced by ~70% in SAT (P < 0.001) in obese vs. control subjects. The degrees of obesity and insulin resistance were both negatively associated with esterification-related parameters and positively with FAO (P < 0.05). Trimetazidine increased skeletal muscle FA esterification (P < 0.01) and mildly upregulated glucose phosphorylation (P = 0.066). Our data suggest that human obesity is characterized by a defect in tissue FA storage capability, which is accompanied by a (potentially compensatory) elevation in skeletal muscle FAO; trimetazidine diverted FA from oxidative to nonoxidative pathways and provoked an initial activation of glucose metabolism in skeletal muscle.     

The role for decorin in delayed-type hypersensitivity

Decorin, a small leucine-rich proteoglycan, regulates extracellular matrix organization, growth factor-mediated signaling, and cell growth. Because decorin may directly modulate immune responses, we investigated its role in a mouse model of contact allergy (oxazolone-mediated delayed-type hypersensitivity [DTH]) in decorin-deficient (Dcn(-/-)) and wild-type mice. Dcn(-/-) mice showed a reduced ear swelling 24 h after oxazolone treatment with a concurrent attenuation of leukocyte infiltration. These findings were corroborated by reduced glucose metabolism, as determined by (18)fluordeoxyglucose uptake in positron emission tomography scans. Unexpectedly, polymorphonuclear leukocyte numbers in Dcn(-/-) blood vessels were significantly increased and accompanied by large numbers of flattened leukocytes adherent to the endothelium. Intravital microscopy and flow chamber and static adhesion assays confirmed increased adhesion and reduced transmigration of Dcn(-/-) leukocytes. Circulating blood neutrophil numbers were significantly increased in Dcn(-/-) mice 24 h after DTH elicitation, but they were only moderately increased in wild-type mice. Expression of the proinflammatory cytokine TNF-α was reduced, whereas syndecan-1 and ICAM-1 were overexpressed in inflamed ears of Dcn(-/-) mice, indicating that these adhesion molecules could be responsible for increased leukocyte adhesion. Decorin treatment of endothelial cells increased tyrosine phosphorylation and reduced syndecan-1 expression. Notably, absence of syndecan-1 in a genetic background lacking decorin rescued the attenuated DTH phenotype of Dcn(-/-) mice. Collectively, these results implicated a role for decorin in mediating DTH responses by influencing polymorphonuclear leukocyte attachment to the endothelium. This occurs via two nonmutually exclusive mechanisms that involve a direct antiadhesive effect on polymorphonuclear leukocytes and a negative regulation of ICAM-1 and syndecan-1 expression.