Circadian time series proteomics reveals daily dynamics in cartilage physiology.

OBJECTIVE: Cartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue. METHODS: We investigated daily changes of protein abundance in mouse femoral head articular cartilage by performing a 48-h time-series LC-MS/MS analysis. RESULTS: Out of the 1,177 proteins we identified across all time points, 145 proteins showed rhythmic changes in their abundance within the femoral head cartilage. Among these were molecules that have been implicated in key cartilage functions, including CTGF, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent functions. Analysis of published cartilage proteomics datasets revealed that a significant portion of rhythmic proteins were dysregulated in osteoarthritis and/or ageing. CONCLUSIONS: Our circadian proteomics study reveals that articular cartilage is a much more dynamic tissue than previously thought, with chondrocytes driving circadian rhythms not only in gene transcription but also in protein abundance. Our results clearly call for the consideration of circadian timing mechanisms not only in cartilage biology, but also in the pathogenesis, treatment strategies and biomarker detection in osteoarthritis.

Mesencephalic astrocyte-derived neurotropic factor is an important factor in chondrocyte ER homeostasis.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) resident protein that can be secreted due to an imperfect KDEL motif. MANF plays a cytoprotective role in several soft tissues and is upregulated in conditions resulting from intracellular retention of mutant protein, including two skeletal diseases, metaphyseal chondrodysplasia, Schmid type (MCDS) and multiple epiphyseal dysplasia (MED). The role of MANF in skeletal tissue homeostasis is currently unknown. Interestingly, cartilage-specific deletion of Manf in a mouse model of MED resulted in increased disease severity, suggesting its upregulation may be chondroprotective. Treatment of MED chondrocytes with exogenous MANF led to a decrease in the cellular levels of BiP (GRP78), confirming MANF's potential to modulate ER stress responses. However, it did not alleviate the intracellular retention of mutant matrilin-3, suggesting that it is the intracellular MANF that is of importance in the pathobiology of skeletal dysplasias. The Col2Cre-driven deletion of Manf from mouse cartilage resulted in a chondrodysplasia-like phenotype. Interestingly, ablation of MANF in cartilage did not have extracellular consequences but led to an upregulation of several ER-resident chaperones including BiP. This apparent induction of ER stress in turn led to dysregulated chondrocyte apoptosis and decreased proliferation, resulting in reduced long bone growth. We have previously shown that ER stress is an underlying disease mechanism for several skeletal dysplasias. The cartilage-specific deletion of Manf described in this study phenocopies our previously published chondrodysplasia models, further confirming that ER stress itself is sufficient to disrupt skeletal growth and thus represents a potential therapeutic target.

Paradoxical roles of ATF6α and ATF6ß in modulating disease severity caused by mutations in collagen X.

Whilst the role of ATF6α in modulating the unfolded protein response (UPR) has been well documented, the function of its paralogue ATF6ß is less well understood. Using knockdown in cell culture and gene ablation in mice we have directly compared the roles of ATF6α & ß in responding to the increased ER stress induced by mutant forms of type X collagen that cause the ER stress-associated metaphyseal chondrodysplasia type Schmid (MCDS). ATF6α more efficiently deals with the disease-associated ER stress in the absence of ATF6ß and conversely, ATF6ß is less effective in the absence of ATF6α. Furthermore, disease severity in vivo is increased by ATF6α ablation and decreased by ATF6ß ablation. In addition, novel functions for each paralogue are described including an ATF6ß-specific role in controlling growth plate chondrocyte proliferation. The clear demonstration of the intimate relationship of the two ATF6 isoforms and how ATF6ß can moderate the activity of ATF6α and vice versa is of great significance for understanding the UPR mechanism. The activities of both ATF6 isoforms and their separate roles need consideration when deciding how to target increased ER stress as a means of treating MCDS and other ER stress-associated diseases.

Gene expression changes in damaged osteoarthritic cartilage identify a signature of non-chondrogenic and mechanical responses.

OBJECTIVES: Joint degeneration in osteoarthritis (OA) is characterised by damage and loss of articular cartilage. The pattern of loss is consistent with damage occurring only where the mechanical loading is high. We have investigated using RNA-sequencing (RNA-seq) and systems analyses the changes that occur in damaged OA cartilage by comparing it with intact cartilage from the same joint. METHODS: Cartilage was obtained from eight OA patients undergoing total knee replacement. RNA was extracted from cartilage on the damaged distal medial condyle (DMC) and the intact posterior lateral condyle (PLC). RNA-seq was performed to identify differentially expressed genes (DEGs) and systems analyses applied to identify dysregulated pathways. RESULTS: In the damaged OA cartilage, there was decreased expression of chondrogenic genes SOX9, SOX6, COL11A2, COL9A1/2/3, ACAN and HAPLN1; increases in non-chondrogenic genes COL1A1, COMP and FN1; an altered pattern of secreted proteinase expression; but no expression of major inflammatory cytokines. Systems analyses by PhenomeExpress revealed significant sub-networks of DEGs including mitotic cell cycle, Wnt signalling, apoptosis and matrix organisation that were influenced by a core of altered transcription factors (TFs), FOSL1, AHR, E2F1 and FOXM1. CONCLUSIONS: Gene expression changes in damaged cartilage suggested a signature non-chondrogenic response of altered matrix protein and secreted proteinase expression. There was evidence of a damage response in this late OA cartilage, which surprisingly showed features detected experimentally in the early response of cartilage to mechanical overload. PhenomeExpress analysis identified a hub of DEGs linked by a core of four differentially regulated TFs.

Catabolic cytokines disrupt the circadian clock and the expression of clock-controlled genes in cartilage via an NFкB-dependent pathway.

OBJECTIVE: To define how the catabolic cytokines (Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFα)) affect the circadian clock mechanism and the expression of clock-controlled catabolic genes within cartilage, and to identify the downstream pathways linking the cytokines to the molecular clock within chondrocytes. METHODS: Ex vivo cartilage explants were isolated from the Cry1-luc or PER2::LUC clock reporter mice. Clock gene dynamics were monitored in real-time by bioluminescence photon counting. Gene expression changes were studied by qRT-PCR. Functional luc assays were used to study the function of the core Clock/BMAL1 complex in SW-1353 cells. NFкB pathway inhibitor and fluorescence live-imaging of cartilage were performed to study the underlying mechanisms. RESULTS: Exposure to IL-1ß severely disrupted circadian gene expression rhythms in cartilage. This effect was reversed by an anti-inflammatory drug dexamethasone, but not by other clock synchronizing agents. Circadian disruption mediated by IL-1ß was accompanied by disregulated expression of endogenous clock genes and clock-controlled catabolic pathways. Mechanistically, NFкB signalling was involved in the effect of IL-1ß on the cartilage clock in part through functional interference with the core Clock/BMAL1 complex. In contrast, TNFα had little impact on the circadian rhythm and clock gene expression in cartilage. CONCLUSION: In our experimental system (young healthy mouse cartilage), we demonstrate that IL-1ß (but not TNFα) abolishes circadian rhythms in Cry1-luc and PER2::LUC gene expression. These data implicate disruption of the chondrocyte clock as a novel aspect of the catabolic responses of cartilage to pro-inflammatory cytokines, and provide an additional mechanism for how chronic joint inflammation may contribute to osteoarthritis (OA).

Cartilage-specific ablation of XBP1 signaling in mouse results in a chondrodysplasia characterized by reduced chondrocyte proliferation and delayed cartilage maturation and mineralization.

OBJECTIVE: To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage. DESIGN: Xbp1(flox/flox).Col2a1-Cre mice (Xbp1(CartΔEx2)), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation. RESULTS: Xbp1(CartΔEx2) displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1(CartΔEx2) growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1(CartΔEx2) embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1(CartΔEx2) growth plate. IRE1 hyperactivation occurred in Xbp1(CartΔEx2) chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR. CONCLUSION: Our work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease.

Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na+ reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 ìmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na+ current by 52 % and increased the membrane density of Na+ channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 ìmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na+ reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage (AU)

Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes.

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na(+) reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 µmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na(+) current by 52% and increased the membrane density of Na(+) channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 µmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na(+) reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage.

Gene expression in human chondrocytes in late osteoarthritis is changed in both fibrillated and intact cartilage without evidence of generalised chondrocyte hypertrophy.

OBJECTIVES: To investigate changes in gene expression in fibrillated and intact human osteoarthritis (OA) cartilage for evidence of an altered chondrocyte phenotype and hypertrophy. METHODS: Paired osteochondral samples were taken from a high-load site and a low-load site from 25 OA joints and were compared with eight similar paired samples from age-matched controls. Gene expression of key matrix and regulatory genes was analysed by quantitative real-time reverse transcription-polymerase chain reaction on total RNA extracted from the cartilage. RESULTS: There was a major change in chondrocyte gene expression in OA cartilage. SOX9 (38-fold) and aggrecan (4-fold) gene expression were both lower in OA (p<0.001), and collagen I (17-fold) and II (2.5-fold) gene expression were each increased in a subset of OA samples. The major changes in gene expression were similar at the fibrillated high-loaded site and the intact low-loaded site. There was no evidence of a generalised change in OA to proliferative or hypertrophic phenotype as seen in the growth plate, as genes associated with either stage of differentiation were unchanged (PTHrPR), or significantly downregulated (collagen X (14-fold, p<0.002), VEGF (23-fold, p<0.02), BCL-2 (5.6-fold, p<0.001), matrilin-1 (6.5-fold, p<0.001)). In contrast MMP-13 was significantly upregulated in the OA cartilage samples (5.3-fold, p<0.003). CONCLUSIONS: The expression of key chondrocyte genes, including aggrecan and SOX9, was decreased in OA cartilage and the changes were similar in both fibrillated high-loaded and intact low-loaded cartilage on the same joint. However, there was no significant upregulation of type X collagen, and other genes associated with chondrocyte further differentiation and hypertrophy.

The biology of the circadian Ck1epsilon tau mutation in mice and Syrian hamsters: a tale of two species.

The tau mutation in the Syrian hamster resides in the enzyme casein kinase 1 epsilon (CK1epsilon), resulting in a dramatic acceleration of wheel-running activity cycles to about 20 hours. tau also impacts growth, energy, metabolism, feeding behavior, and circadian mechanisms underpinning seasonal timing, causing accelerated reproductive and neuroendocrine responses to photoperiodic changes. Modeling and experimental studies suggest that tau acts as a gain of function on specific residues of PER, consistent with hamster studies showing accelerated degradation of PER in the suprachiasmatic nucleus in the early circadian night. We have created null and tau mutants of Ck1epsilon in mice. Circadian period lengthens in CK1epsilon(/), whereas CK1epsilon(tau/tau) shortens circadian period of behavior in vivo in a manner nearly identical to that of the Syrian hamster. CK1epsilon(tau/tau) also accelerates molecular oscillations in peripheral tissues, demonstrating its global circadian role. CK1epsilon(tau) acts by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Our studies reveal that tau acts as a gain-of-function mutation, to accelerate degradation of PERIOD proteins. tau has consistent effects in both hamsters and mice on the circadian organization of behavior and metabolism, highlighting the global impact of this mutation on mammalian clockwork in brain and periphery.

Activation of extracellular signal-regulated protein kinase 5 downregulates FasL upon osmotic stress.

Extracellular signal-regulated protein kinase (ERK) 5 is a mitogen-activated protein kinase (MAPK) that is activated by dual phosphorylation via a unique MAPK/ERK kinase 5, MEK5. The physiological importance of this signaling cascade is underscored by the early embryonic death caused by the targeted deletion of the erk5 or the mek5 genes in mice. Here, we have found that ERK5 is required for mediating the survival of fibroblasts under basal conditions and in response to sorbitol treatment. Increased Fas ligand (FasL) expression acts as a positive feedback loop to enhance apoptosis of ERK5- or MEK5-deficient cells under conditions of osmotic stress. Compared to wild-type cells, erk5-/- and mek5-/- fibroblasts treated with sorbitol display a reduced protein kinase B (PKB) activity associated with increased Forkhead box O3a (Foxo3a) activity. Based on these results, we conclude that the ERK5 signaling pathway promotes cell survival by downregulating FasL expression via a mechanism that implicates PKB-dependent inhibition of Foxo3a downstream of phosphoinositide 3 kinase.

Transcription factor XBP-1 is expressed during osteoblast differentiation and is transcriptionally regulated by parathyroid hormone (PTH).

X-box-binding protein 1 (XBP-1) is a basic-region leucine zipper protein in the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors involved in different cell-differentiation processes. We have investigated the expression of XBP-1 in differentiating MC3T3-E1 osteoblastic cells. Cultures were treated with ascorbic acid (AA) and beta-glycerophosphate (BGP) to induce differentiation. Under these conditions, the basal transcription of xbp-1 increases at day 2 following induction, peaks at day 5 and decreases thereafter. This result showed that xbp-1 gene is differentially expressed during MC3T3-E1 cell differentiation. Detection of XBP-1 by immunofluorescence at days 0 (control culture without AA and BGP), 8 and 21 showed that the protein has a major cytoplasmic perinuclear location. In addition, xbp-1 is transcriptionally upregulated by parathyroid hormone within 2.5 h of treatment and decreases thereafter.

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis.

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.

Molecular and functional studies of electrogenic Na(+) transport in the distal colon and rectum of young and elderly subjects.

BACKGROUND: Human distal nephron and distal colon both exhibit mineralocorticoid sensitive electrogenic Na(+) absorption and make significant contributions to Na(+) homeostasis. Na(+) resorption in the distal nephron diminishes with age but it is unclear whether a similar change occurs in the distal colon. AIMS: To evaluate the effect of age on expression of apical Na(+) channels and basolateral Na(+), K(+)-ATPase, and on the responsiveness of electrogenic Na(+) absorption to mineralocorticoid stimulation in human distal colon and rectum. MATERIALS AND METHODS: Mucosal biopsies were obtained from healthy sigmoid colon and proximal rectum in "young" (aged 20-40 years) and "old" (aged 70 years or over) patients during routine colonoscopy/flexible sigmoidoscopy. Na(+) channel subunits and Na(+), K(+)-ATPase isoforms were studied at the mRNA level by in situ hybridisation and northern blotting, and at the protein level by immunocytochemistry and western blotting. The mineralocorticoid responsiveness of electrogenic Na(+) absorption was evaluated in the two groups by measuring amiloride sensitive electrical potential difference (PD) in the proximal rectum before and 24 hours after oral administration of 1 mg of fludrocortisone. RESULTS: Na(+) channel subunit and Na(+), K(+)-ATPase isoform expression at the level of mRNA and protein was similar in "young" and "old" patients. Both basal and the fludrocortisone stimulated amiloride sensitive rectal PDs were similar in the two groups. CONCLUSIONS: In contrast with the distal nephron, mineralocorticoid sensitive electrogenic Na(+) absorption in the human distal colon does not diminish with age, and may be particularly important in maintaining Na(+) homeostasis in the elderly.

Segmental variability of ENaC subunit expression in rat colon during dietary sodium depletion.

In rat distal colon, aldosterone has little effect on Na(+) channel (ENaC) alpha-subunit levels but increases the expression of the beta- and gamma-subunits and stimulates electrogenic Na(+) transport. By contrast, the molecular basis of aldosterone's inability to stimulate electrogenic Na(+) transport in the proximal colon is unclear. We therefore compared the effects of hyperaldosteronism secondary to 10 days dietary Na(+) depletion on ENaC subunit expression in rat proximal and distal colon. Northern analyses revealed appreciable and similar levels of alpha-subunit mRNA throughout the colon in control and Na(+)-depleted animals. By contrast, Na(+) depletion substantially enhanced beta-subunit mRNA expression in the distal colon, but had no effect on the low expression levels of beta-subunit mRNA in the proximal colon. Expression of the gamma-subunit, evaluated by PCR, was also restricted to the distal colon of Na(+)-depleted animals. Western analyses demonstrated similar levels of alpha-subunit protein in the proximal and distal colon of both groups of animals, whereas beta-subunit and gamma-subunit proteins were detected solely or predominantly in the distal colon of the Na(+)-depleted animals. Immunocytochemistry confirmed that significant levels of all three subunit proteins only occurred in the apical membrane of surface cells in the distal colon of Na(+)-depleted animals. Our findings are consistent with previous studies demonstrating that aldosterone stimulates electrogenic Na(+) transport in rat distal colon by increasing the expression of beta- and gamma-subunit mRNA and protein, and thus the amount of functional heteromeric ENaC protein in the apical domain. They also show that aldosterone is incapable of stimulating electrogenic Na(+) transport in rat proximal colon (despite the presence of alpha-subunit mRNA and protein) because of its inability to enhance beta- and gamma-subunit expression in this segment.

The involvement of matrix glycoproteins in vascular calcification and fibrosis: an immunohistochemical study.

Calcification and fibrointimal proliferation are associated with advanced complicated atherosclerosis in large arteries but may also occur in smaller vessels, resulting in ischaemic tissue necrosis. This study investigates whether the mechanisms of calcification and intimal fibrosis are similar in vessels of different sizes. The localization of osteopontin (OPN), matrix Gla protein (MGP), thrombospondin-1 (TSP-1), and cartilage oligomeric matrix protein (COMP) was investigated in three types of human vascular lesions: atherosclerosis, chronic vascular rejection (CVR) in renal allografts, and calcific uraemic arteriolopathy (calciphylaxis). These lesions were chosen as they affect different sized blood vessels and they exhibit a fibroproliferative intimal reaction, with or without calcification, resulting in luminal obliteration and ischaemic complications. OPN, MGP, TSP-1, and COMP were not detected in normal blood vessels. However, OPN and MGP were expressed at sites of calcification within atherosclerotic lesions and in microvessels in calciphylaxis, suggesting that calcification in different sized vessels may occur by a common mechanism. These proteins were not detected in areas of fibrointimal proliferation. In contrast, TSP-1 was localized primarily within the fibrous tissue of atherosclerotic lesions and was also expressed in the expanded fibrous intima of arteries showing CVR. COMP was localized primarily within the fibrous tissue under the lipid core of the majority of advanced atherosclerotic lesions. TSP-1 and COMP were also detected in areas of microcalcification in atherosclerotic lesions and TSP-1 was detected adjacent to areas of calcification in calciphylaxis. However, neither TSP-1 nor COMP was localized to calcific foci within these lesions. The localization of OPN, MGP, TSP-1, and COMP to pathological, but not normal arterial intima supports a pathogenetic role for these proteins in the development of vascular fibrosis and calcification. Modulation of their production and activity may offer a novel approach to the therapy of a number of vascular diseases.

Molecular, biochemical and functional analysis of a novel and developmentally important fibrillar collagen (Hcol-I) in hydra.

The body wall of hydra (a member of the phylum Cnidaria) is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Previous studies have established that cell-ECM interactions are important for morphogenesis and cell differentiation in this simple metazoan. The ECM of hydra is particularly interesting because it represents a primordial form of matrix. Despite progress in our understanding of hydra ECM, we still know little about the nature of hydra collagens. In the current study we provide a molecular, biochemical and functional analysis of a hydra fibrillar collagen that has similarity to vertebrate type I and type II collagens. This fibrillar collagen has been named hydra collagen-I (Hcol-I) because of its structure and because it is the first ECM collagen to be identified in hydra. It represents a novel member of the collagen family. Similar to vertebrate type I and II collagens, Hcol-I contains an N-terminal propeptide-like domain, a triple helical domain containing typical Gly-X-Y repeats and a C-terminal propeptide domain. The overall identity to vertebrate fibrillar collagens is about 30%, while the identity of the C-terminal propeptide domain is 50%. Because the N-terminal propeptide domain is retained after post-translational processing, Hcol-I does not form thick fibers as seen in vertebrates. This was confirmed using transmission electron microscopy to study rotary shadow images of purified Hcol-I. In addition, absence of crucial lysine residues and an overall reduction in proline content, results in reduced crosslinking of fibrils and increased flexibility of the molecule, respectively. These structural changes in Hcol-I help to explain the flexible properties of hydra ECM. Immunocytochemical studies indicate that Hcol-I forms the 10 nm fibrils that comprise the majority of molecules in the central fibrous zone of hydra ECM. The central fibrous zone resides between the two subepithelial zones where hydra laminin is localized. While previous studies have shown that basal lamina components like laminin are expressed by the endoderm, in situ hybridisation studies show that Hcol-I mRNA expression is restricted to the ectoderm. Hcol-I expression is upregulated during head regeneration, and antisense studies using thio-oligonucleotides demonstrated that blocking the translation of Hcol-I leads to a reversible inhibition of head morphogenesis during this regenerative process. Taken in total, the data presented in this study indicate that Hcol-I is required for morphogensis in hydra and represents a novel fibrillar collagen whose structural characteristics help to explain the unique biophysical properties of hydra ECM. Interestingly, the structure of Hcol-I mimics what is seen in Ehlers-Danlos syndrome type VII in humans; an inherited pathological condition that leads to joint and skin abnormalities. Hcol-I therefore illustrates an adaptive trait in which the normal physiological situation in hydra translates into a pathological condition in humans.

Characterization of hydra type IV collagen. Type IV collagen is essential for head regeneration and its expression is up-regulated upon exposure to glucose.

Hydra vulgaris mesoglea is a primitive basement membrane that also exhibits some features of an interstitial matrix. We have characterized cDNAs that encode the full-length hydra alpha1(IV) chain. The 5169-base pair transcript encodes a protein of 1723 amino acids, including an interrupted 1455-residue collagenous domain and a 228-residue C-terminal noncollagenous domain. N-terminal sequence analyses of collagen IV peptides suggest the molecule is homotrimeric. Denatured hydra type IV collagen protein occurs as dimers and higher order aggregates held together by nonreducible cross-links. Hydra collagen IV exhibits no functional evidence for the presence of a 7 S domain. Type IV collagen is expressed by the ectoderm along the entire longitudinal axis of the animal but is most intense at the base of the tentacles at the site of battery cell transdifferentiation. Antisense studies show that inhibition of collagen IV translation causes a blockage in head regeneration, indicating its importance in normal hydra development. Exposure of adult hydra to 15 mm glucose resulted in up-regulation of type IV collagen mRNA levels within 48 h and significant thickening of the mesoglea within 14 days, suggesting that basement membrane thickening seen in diabetes may be, in evolutionary terms, an ancient glucose-mediated response.

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.