Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. Methods: Bovine calf articular cartilage discs or isolated cells were used in I of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from similar to25 mug/cm(2)/day on days 0-1 to similar to3 mug/cm(2)/day on days 5-9), while increasing in monolayer culture (from similar to1 pg/cell/day on days 0-1 to similar to7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching similar to2 mug/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

Tailoring secretion of proteoglycan 4 (PRG4) in tissue-engineered cartilage

Articular cartilage provides a low-friction surface for joint articulation, with boundary lubrication facilitated by proteoglycan 4 (PRG4), which is secreted by chondrocytes of the superficial zone. Chondrocytes from different zones are phenotypically distinct, and their phenotypes in vitro are influenced by the system in which they are cultured. We hypothesized that culturing cells from the superficial (S) zone in two-dimensional monolayer or three-dimensional alginate would affect their synthesis of PRG4, and that subsequently seeding them atop alginate-recovered cells from the middle/ deep (M) zone in various proportions would result in tissue-engineered constructs with varying levels of PRG4 secretion and matrix accumulation. During monolayer culture, S cells retained their PRG4-secreting phenotype, whereas in alginate culture the percentage of cells secreting PRG4 decreased with time. Constructs formed with increasing percentages of S cells decreased in thickness and matrix accumulation, depending on both the culture conditions before construct formation and the S-cell density. PRG4-secreting cells were localized to the S-cell seeded construct surface, with secretion rates of 0.1–4 pg/cell/day or 0.1–1 pg/cell/day for constructs formed with monolayer-recovered or alginate-recovered S cells, respectively. Tailoring secretion of PRG4 in cartilage constructs may be useful for enhancing low-friction properties at the articular surface, while maintaining other surfaces free of PRG4 for enhancing integration with surrounding tissues.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor α (TNFα) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFα is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasmamembrane, and we investigated a possible role for lipid rafts in TNFα trafficking and secretion. TNFα surface delivery and secretion were found to be cholesterol- dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasmamembrane, particularly on filopodia. Imaging the early stages of TNFα surface distribution revealed these puncta to be the initial points of TNFα delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

A role for the phagosome in cytokine secretion

Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor α (TNFα) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNFα and expands the membrane for phagocytosis.

The FAM deubiquitylating enzyme localizes to multiple points of protein trafficking in epithelia, where it associates with E-cadherin and β-catenin

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, β-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with β-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with β-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with β-catenin and E-cadherin from a higher molecular weight complex (~500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, β-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (~2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased β-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and β-catenin during trafficking to the plasma membrane.

Flightless, secreted through a late endosome/lysosome pathway, binds LPS and dampens cytokine secretion.

Flightless (Flii) is upregulated in response to wounding and has been shown to function in wound closure and scarring. In macrophages intracellular Flii negatively modulates TLR signalling and dampens cytokine production. We now show that Flii is constitutively secreted from macrophages and fibroblasts and is present in human plasma. Secretion from fibroblasts is upregulated in response to scratch wounding and LPS-activated macrophages also temporally upregulate their secretion of Flii. Using siRNA, wild-type and mutant proteins we show that Flii is secreted via a late endosomal/lysosomal pathway that is regulated by Rab7 and Stx11. Flii contains 11 leucine rich repeat (LRR) domains in its N-terminus that have nearly 50% similarity to those in the extracellular pathogen binding portion of Toll-like receptor 4 (TLR4). We show secreted Flii can also bind LPS and has the ability to alter macrophage activation. LPS activation of macrophages in Flii depleted conditioned media leads to enhanced macrophage activation and increased TNF secretion compared to cells activated in the presence of Flii. These results show secreted Flii binds to LPS and in doing so alters macrophage activation and cytokine secretion, suggesting that like the intracellular pool of Flii, secreted Flii also has the ability to alter inflammation.

Aromatic l-amino acid decarboxylase : histochemical localization in rat kidney and lack of effect of dietary potassium or sodium loading on enzyme distribution

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.

Expression of PTRF in PC3 cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Lysosomal secretion of Flightless I upon injury has the potential to alter inflammation

Intracellular Flightless I (Flii), a gelsolin family member, has been found to have roles modulating actin regulation, transcriptional regulation and inflammation. In vivo Flii can regulate wound healing responses. We have recently shown that a pool of Flii is secreted by fibroblasts and macrophages, cells typically found in wounds, and its secretion can be upregulated upon wounding. We show that secreted Flii can bind to the bacterial cell wall component lipopolysaccharide and has the potential to regulate inflammation. We now show that secreted Flii is present in both acute and chronic wound fluid.

Intracellular trafficking and secretion of inflammatory cytokines

The secretion of cytokines by immune cells plays a significant role in determining the course of an inflammatory response. The levels and timing of each cytokine released are critical for mounting an effective but confined response, whereas excessive or dysregulated inflammation contributes to many diseases. Cytokines are both culprits and targets for effective treatments in some diseases. The multiple points and mechanisms that have evolved for cellular control of cytokine secretion highlight the potency of these mediators and the fine tuning required to manage inflammation. Cytokine production in cells is regulated by cell signaling, and at mRNA and protein synthesis levels. Thereafter, the intracellular transport pathways and molecular trafficking machinery have intricate and essential roles in dictating the release and activity of cytokines. The trafficking machinery and secretory (exocytic) pathways are complex and highly regulated in many cells, involving specialized membranes, molecules and organelles that enable these cells to deliver cytokines to often-distinct areas of the cell surface, in a timely manner. This review provides an overview of secretory pathways - both conventional and unconventional - and key families of trafficking machinery. The prevailing knowledge about the trafficking and secretion of a number of individual cytokines is also summarized. In conclusion, we present emerging concepts about the functional plasticity of secretory pathways and their modulation for controlling cytokines and inflammation.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

Three native freshwater crayfish Cherax species are farmed in Australia namely; Redclaw (Cherax quadricarinatus), Marron (C. tenuimanus), and Yabby (C. destructor). Lack of appropriate data on specific nutrient requirements for each of these species, however, has constrained development of specific formulated diets and hence current use of over-formulated feeds or expensive marine shrimp feeds, limit their profitability. A number of studies have investigated nutritional requirements in redclaw that have focused on replacing expensive fish meal in formulated feeds with non-protein, less expensive substitutes including plant based ingredients. Confirmation that freshwater crayfish possess endogenous cellulase genes, suggests their potential ability to utilize complex carbohydrates like cellulose as nutrient sources in their diet. To date, studies have been limited to only C. quadricarinatus and C. destructor and no studies have compared the relative ability of each species to utilize soluble cellulose in their diets. Individual feeding trials of late-juveniles of each species were conducted separately in an automated recirculating culture system over 12 week cycles. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch. Water temperature, conductivity and pH were maintained at constant and optimum levels for each species. Animals were fed at 3% of their body weight twice daily and wet body weight was recorded bi-weekly. At the end of experiment, all animals were harvested, measured and midgut gland extracts assayed for alpha-amylase, total protease and cellulase activity levels. After the trial period, redclaw fed with RD showed significantly higher (p<0.05) specific growth rate (SGR) compare with animals fed the TD while SGR of marron and yabby fed the two diets were not significantly different (p<0.05). Cellulase expression levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD. Amylase and protease activity in all three species were significantly higher in the animals fed with RD (Table 1). These results indicate that test animals of all species can utilize starch better than dietary soluble cellulose in their diet and inclusion of 20% soluble cellulose in diets does not appear to have any significant negative effect on their growth rate but survival was impacted in C. quadricarinatus while not in C. tenuimanus or C. destructor.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

The current study evaluated the effect of soluble dietary cellulose on growth, survival and digestive enzyme activity in three endemic, Australian freshwater crayfish species (redclaw: Cherax quadricarinatus, marron: C. tenuimanus, yabby: C. destructor). Separate individual feeding trials were conducted for late-stage juveniles from each species in an automated recirculating freshwater, culture system. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch, over a 12 week period. Redclaw fed with RD showed significantly higher (p<0.05) specific growth rates (SGR) compared with animals fed the TD, while SGR of marron and yabby fed the two diets were not significantly different. Expressed cellulase activity levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD (p<0.05). Amylase and protease activity in all three species were significantly higher in the animals fed with RD (p<0.05). These results indicate that test animals of all three species appear to utilize starch more efficiently than soluble dietary cellulose in their diet. The inclusion of 20% soluble cellulose in diets did not appear, however, to have a significant negative effect on growth rates.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5 L (15–25 FPU/g glucan) was in the following order: glycerol/HCl > HCl > H2SO4 > NaOH, with the glycerol system achieving ∼100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6–6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2–6 times that of acid substrates. However, glucan conversions (15–60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10–30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.