Prolyl and lysyl hydroxylases in collagen synthesis.

Collagens are the most abundant proteins in the extracellular matrix. They provide a framework to build organs and tissues and give structural support to make them resistant to mechanical load and forces. Several intra- and extracellular modifications are needed to make functional collagen molecules, intracellular post-translational modifications of proline and lysine residues having key roles in this. In this article, we provide a review on the enzymes responsible for the proline and lysine modifications, that is collagen prolyl 4-hydroxylases, 3-hydroxylases and lysyl hydroxylases, and discuss their biological functions and involvement in diseases.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

Inflammatory pattern of the infrapatellar fat pad in dogs with canine cruciate ligament disease.

BACKGROUND: Despite the importance of inflammation during the pathogenesis of cranial cruciate ligament disease (CCLD) in dogs and despite the latest knowledge suggesting a significant role of adipose tissue in osteoarthritis, the infrapatellar fat pad (IFP) was up to now mostly disregarded in veterinary investigations. In the present study, the inflammatory activity of the IFP, the main adipose structure within the stifle joint, was thoroughly investigated to evaluate its potential impact in the pathogenesis of this common disease of our canine companions. Samples of IFP, subcutaneous adipose tissue (ScAT) of the thigh and synovial fluid in both diseased (n = 36) and healthy control (n = 23) dogs were tested for their immune cell composition but also for interleukins (IL-1ß, IL-6, IL-8, IL-10), degradative enzymes (MMP-1, MMP-3, MMP-13, TIMP-2, iNOS) and adipokines (leptin and adiponectin). Characterization of the immune cell composition was ascertained by fluorescence activated cell sorting. Gene expression and protein release of the inflammatory markers was determined by real RT-qPCR and ELISA. RESULTS: IFPs of dogs with CCLD had a significantly increased immune cell count with T cells (CD3) as the most abundant immune cells. T cells and macrophages (CD14) were significantly increased compared to healthy controls or corresponding ScAT. In addition, IFPs of dogs with CCLD demonstrated a significant increase on gene as well as protein level of multiple inflammatory indicators (IL-1ß, IL-6, MMP-1, MMP-13) compared to the other tissues. TNFα was only increased on gene expression. Adipokine analysis showed higher secretion of adiponectin and lower leptin secretion in IFP from dogs with CCLD than from controls. In the synovial fluid from dogs with CCLD concentrations of IL-1ß, MMP-1, MMP-13 as well as leptin were significantly increased compared to the synovial fluid from healthy control dogs. CONCLUSIONS: The present study indicates that the IFP is a potential contributory factor in the pathogenesis of CCLD, due to its inflammatory phenotype and the proximity within the stifle joint. To determine the extent of this possible inter-relationship, further studies need to be undertaken.

Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number.

Elevated basal serum tryptase levels are present in 4-6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase-encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.

Treatment and outcomes in necrotising autoimmune myopathy: An Australian perspective.

Necrotising Autoimmune Myopathy (NAM) presents as a subacute proximal myopathy with high creatine kinase levels. It is associated with statin exposure, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibody, connective tissue diseases, signal recognition particle (SRP) antibody and malignancy. This case series presents our Western Australian NAM patient cohort: comparing the subgroup presentations, biopsy appearance and treatment outcomes. We retrospectively collected data on patients diagnosed with NAM at the Western Australian Neuroscience Research Institute between the years 2000 and 2015. We identified 20 patients with Necrotising Autoimmune Myopathy: 14 with anti-HMGCR antibodies; two with anti-SRP antibodies; three with connective tissue disease; two as yet unspecified. Median creatine kinase level was 6047units/L (range 1000-17000). The statin naïve patients with HMGCR antibodies and patients with SRP antibodies were the most severely affected subgroups, with higher creatine kinase levels, and were more resistant to immunotherapy. Two or more immunotherapy agents were required in 90%; eight patients required IVIG and rituximab. Steroid weaning commonly precipitated relapses. Four patients had complete remission, and the remaining patients still require immunotherapy. Necrotising Autoimmune Myopathy is a potentially treatable myopathy, which can be precipitated by statin therapy and requires early, aggressive immunotherapy, usually requiring multiple steroid sparing agents for successful steroid weaning.

Cellular mechanisms of tissue fibrosis. 8. Current and future drug targets in fibrosis: focus on Rho GTPase-regulated gene transcription.

Tissue fibrosis occurs with excessive extracellular matrix deposition from myofibroblasts, resulting in tissue scarring and inflammation. It is driven by multiple mediators, such as the G protein-coupled receptor ligands lysophosphatidic acid and endothelin, as well as signaling by transforming growth factor-ß, connective tissue growth factor, and integrins. Fibrosis contributes to 45% of deaths in the developed world. As current therapeutic options for tissue fibrosis are limited and organ transplantation is the only effective treatment for end-stage disease, there is an imminent need for efficacious antifibrotic therapies. This review discusses the various molecular pathways involved in fibrosis. It highlights the Rho GTPase signaling pathway and its downstream gene transcription output through myocardin-related transcription factor and serum response factor as a convergence point for targeting this complex set of diseases.

Safety and efficacy of mizoribine in patients with connective tissue diseases other than rheumatoid arthritis.

The objective of this study was to examine the safety and efficacy of mizoribine (MZR), an inhibitor of inosine monophosphate dehydrogenase, in patients with connective tissue diseases (CTDs) other than rheumatoid arthritis. We identified all patients who had ever been treated with MZR for CTDs at our institution during the period from January 2001 to May 2011. A retrospective review of medical records was performed to evaluate safety and efficacy of MZR. A total of 63 patients (13 induction and 50 maintenance therapy with MZR) were included. During 70.2 patient-years of follow-up, only one patient required discontinuation of MZR due to an adverse event. Doses of PSL were significantly decreased at last follow-up in both the induction (45.2 ± 15.6 vs. 8.4 ± 5.7 mg/day, p < 0.01) and the maintenance group (12.4 ± 7.6 vs. 9.3 ± 6.4 mg/day, p < 0.01). MZR appears to be a safe and well-tolerated steroid-sparing agent in patients with CTDs.

Significant association between CYP3A5 polymorphism and blood concentration of tacrolimus in patients with connective tissue diseases.

Although the association between CYP3A5 polymorphism and blood concentration of tacrolimus (TAC) in patients with solid organ transplantation was established, whether the association is also true in patients with connective tissue disease (CTD) who usually receive small amount of TAC is uncertain. Here, we performed a quantitative linear regression analysis to address the association between CYP3A5 and blood TAC concentration in patients with CTD. A total of 72 patients with CTD were recruited in the current study and genotyped for rs776746 in CYP3A5, which showed strong association with TAC concentration in patients with solid organ transplantation. The blood trough concentration of TAC after taking 3 mg per day was retrospectively obtained for each patient. As a result, allele A of rs776746 showed a significant association with a decreasing blood concentration of TAC (P=0.0038). Those who are carrying at least one copy of the A allele displayed decreased mean concentration of TAC by 31.0% compared with subjects with GG genotype. Rs776746 is associated with concentrations of TAC in patients with CTD.

The expression of matrix metalloproteinases in placental tissue depends on the severity of undifferentiated connective tissue dysplasia.

Immunohistochemical study of MMP-2 and MMP-9 expression in placental tissue of pregnant patients with undifferentiated connective tissue dysplasia of different severity showed that more severe condition was associated with higher expression of these MMP, this underlying the development of pregnancy and labor complications. The most pronounced elevation of the studied MMP levels was found in the basal plate decidual cells in women with undifferentiated connective tissue dysplasia of more than 18 score.

Human genetic disorders caused by mutations in genes encoding biosynthetic enzymes for sulfated glycosaminoglycans.

A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs.

Relevance of the inner mitochondrial membrane enzyme F1F0-ATPase as an autoantigen in autoimmune liver disorders.

BACKGROUND AND AIMS: Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody (AMA) negative sera from patients with primary biliary cirrhosis (PBC) has been shown to contain parts of the five F(1)-ATPase subunits α, ß, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC. METHODS: Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis (AIH), 35 patients with primary sclerosing cholangitis (PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases (IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F(1)-ATPase-subunits α-δ were recombinantly expressed in Escherichia coli, purified and applied to ELISA and Western blotting. RESULTS: In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F(1)-subunits α-δ. The ß- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-ß- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-ß and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the ß- and only 6% to the γ-subunit. Sera from healthy blood donors were negative. CONCLUSIONS: Antibodies to the ß- and γ-subunits of F(1)-ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver.

Missense mutations that cause Bruck syndrome affect enzymatic activity, folding, and oligomerization of lysyl hydroxylase 2.

Bruck syndrome is a rare autosomal recessive connective tissue disorder characterized by fragile bones, joint contractures, scoliosis, and osteoporosis. The telopeptides of bone collagen I are underhydroxylated in these patients, leading to abnormal collagen cross-linking. Three point mutations in lysyl hydroxylase (LH) 2, the enzyme responsible for the hydroxylation of collagen telopeptides, have been identified in Bruck syndrome. As none of them affects the residues known to be critical for LH activity, we studied their consequences at the molecular level by analyzing the folding and catalytic properties of the corresponding mutant recombinant polypeptides. Folding and oligomerization of the R594H and G597V mutants were abnormal, and their activity was reduced by >95% relative to the wild type. The T604I mutation did not affect the folding properties, although the mutant retained only approximately 8% activity under standard assay conditions. As the reduced activity was caused by a 10-fold increase in the K(m) for 2-oxoglutarate, the mutation interferes with binding of this cosubstrate. In the presence of a saturating 2-oxoglutarate concentration, the activity of the T604I mutant was approximately 30% of that of the wild type. However, the T604I mutant did not generate detectable amounts of hydroxylysine in the N-terminal telopeptide of a recombinant procollagen I chain when coexpressed in insect cells. The low activity of the mutant LH2 polypeptides is in accordance with the markedly reduced extent of collagen telopeptide hydroxylation in Bruck syndrome, with consequent changes in the cross-linking of collagen fibrils and severe abnormalities in the skeletal structures.

Heterogeneous pathogenic processes of thrombotic microangiopathies in patients with connective tissue diseases.

To clarify the pathogenic processes of thrombotic microangiopathies (TMAs) in patients with connective tissue disease (CTD), we analysed clinical characteristics and plasma ADAMTS13 levels in 127 patients with CTD-TMAs, including patients with systemic lupus erythematosus (SLE), systemic sclerosis, polymyositis/dermatomyositis, and rheumatoid arthritis (RA), and 64 patients with acquired idiopathic thrombotic thrombocytopenic purpura (ai-TTP). Plasma levels of ADAMTS13 activity, antigen, and inhibitors were determined by enzyme immunoassays. IgG type anti-ADAMTS13 antibodies were also detected by immunoblots using purified ADAMTS13. ADAMTS13 activity was significantly decreased in CTD-TMAs, regardless of the underlying disease, but the frequency of severe deficiency (defined as <0.5% of normal) was lower in CTD-TMA patients than in ai-TTP patients (16.5% vs. 70.3%, p < 0.01). Severe deficiency of ADAMTS13 activity was predominantly detected in patients with RA- and SLE-TMAs, and was closely associated with the presence of anti-ADAMTS13 IgG antibodies. CTD-TMA patients with severe deficiency of ADAMTS13 activity appeared to have lower platelet counts and better therapeutic outcomes. At least two phenotypic TMAs occur in patients with CTDs: a minor population with deficient ADAMTS13 activity caused by neutralising autoantibodies, and a major population with normal or moderately reduced activity. Classifying CTD-TMAs by ADAMTS13 activity may be useful in predicting the clinical course and therapeutic outcomes, as patients with moderately reduced activity are likely to have more prominent renal impairment and poor prognoses.

Will Jill come tumbling after? The case for a JAK2-type mutation as a prequel to the connective tissue disorders.

The JAK2 [V617F] mutation has recently been recognised as critical to the pathogenesis of the myeloproliferative disorders (MPDs). Thus, a common mutation affecting a haematopoietic precursor stem cell is capable of giving rise to diverse clinical phenotypes. In this hypothesis paper, we propose that a similar mutation affecting a stem cell precursor, most likely of the B cell lineage, could underlie the development of the connective tissue disorders which could be regarded as "lymphoproliferative" disorders. Consistent with this hypothesis is the observation that there are similarities between the myeloproliferative disorders and the connective tissue disorders in terms of their biological behaviour. Diseases within each family can transform into each other and sometimes into haematological malignancies (most often B cell origin non-Hodgkins lymphoma for the connective tissue disorders and acute myeloid leukemia for the myeloproliferative disorders). The timecourse for development of the connective tissue disorders involves a long latent period when autoantibodies are present (anti-CCP and ANA) possibly reflecting production by a B cell clone. A similar time-dependent increase in clonal dominance has been described in erythroblastic clones taken from the bone marrow of polycythemia vera patients, long before the onset of clinical disease. Evidence of B cell clonality has been described in bone marrow samples from rheumatoid arthritis patients and from glandular biopsies from those with Sjogren's syndrome. Moreover, pseudofollicles containing activated B cells are features of rheumatoid synovial membrane and have also recently been described in subchondral bone where they are associated with macrophages, T cells and osteoclasts. The success of B cell depletion therapy in rheumatoid arthritis and systemic lupus erythematosus is also strong circumstantial evidence for this hypothesis.

Telomerase activity in connective tissue diseases: elevated in rheumatoid arthritis, but markedly decreased in systemic sclerosis.

Telomerase is a reverse transcriptase enzyme contributing to the maintenance of the telomeric structure by adding telomere repeat sequences to chromosomal ends, thus compensating for its shortening. Telomerase activity which is common in cancers and human germ line tissue, may also be increased, although to a lesser extent, in systemic autoimmune diseases. We aimed to evaluate telomerase activity in a group of Turkish patients with various connective tissue diseases. In this cross sectional study, 19 patients with systemic sclerosis (SSc), 15 with systemic lupus erythematosus (SLE), 10 with rheumatoid arthritis (RA) and 14 with primary Sjögren's syndrome (pSjS) were studied. As the control group, 29 healthy subjects were also included. Human telomerase-specific reverse transcriptase (hTERT) was measured in peripheral blood lymphocytes, using online real-time reverse-transcriptase polymerase chain reaction (PCR). We also investigated if hTERT values in each patient group were correlated with clinical parameters and disease activity. Highest hTERT values were observed in RA group (21.24 +/- 28.54), followed by SLE (13.38 +/- 26.05) and pSjS (11.73 +/- 10.59) groups. Only hTERT values in RA group was significantly higher than the healthy control group (7.62 +/- 4.21) (p < 0.05). Interestingly, hTERT values in SSc were very low (2.09 +/- 3.18), even less than the healthy control group. In consistent with previous studies, telomerase activity was increased in SLE and RA. Very low telomerase activity in SSc group was rather surprising. Since existing telomerase data in SSc was limited and telomere shortening was previously reported in SSc, our finding of low telomerase activity in SSc group deserves relevant discussion and further studies.

Human xylosyltransferases in health and disease.

The xylosyltransferases I and II (XT-I, XT-II, EC 2.4.2.26) catalyze the transfer of xylose from UDP-xylose to selected serine residues in the proteoglycan core protein, which is the initial and ratelimiting step in glycosaminoglycan biosynthesis. Both xylosyltransferases are Golgi-resident enzymes and transfer xylose to similar core proteins acceptors. XT-I and XT-II are differentially expressed in cell types and tissues, although the reason for the existence of two xylosyltransferase isoforms in all higher organisms remains elusive. Serum xylosyltransferase activity was found to be a biochemical marker for the assessment of disease activity in systemic sclerosis and for the diagnosis of fibrotic remodeling processes. Furthermore, sequence variations in the XT-I and XT-II coding genes were identified as risk factors for diabetic nephropathy, osteoarthritis or pseudoxanthoma elasticum. These findings point to the important role of the xylosyltransferases as disease modifiers in pathologies which are characterized by an altered proteoglycan metabolism. The present review discusses recent advances in mammalian xylosyltransferases and the impact of xylosyltransferases in proteoglycan-associated diseases.

Molecular characterisation of six patients with prolidase deficiency: identification of the first small duplication in the prolidase gene and of a mutation generating symptomatic and asymptomatic outcomes within the same family.

Prolidase deficiency (PD) is a rare autosomal recessive connective tissue disorder caused by mutations in the prolidase gene. The PD patients show a wide range of clinical outcomes characterised mainly by intractable skin ulcers, mental retardation and recurrent respiratory infections. Here we describe five different PEPD mutations in six European patients. We identified two new PEPD mutant alleles: a 13 bp duplication in exon 8, which is the first reported duplication in the prolidase gene and a point mutation resulting in a change in amino acid E412, a highly conserved residue among different species. The E412K substitution is responsible for the first reported phenotypic variability within a family with severe and asymptomatic outcomes.

ADAMTS proteinases: potential therapeutic targets?

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases have been implicated in a number of connective tissue pathologies including Ehlers-Danlos syndrome type VII C, Weill-Marchesani syndrome, encephalomyelitis, and arthritis. These proteinases therefore represent potential therapeutic targets for the treatment of such conditions. The synthesis and activity of ADAMTS proteinases is regulated at multiple levels: transcription, RNA splicing, translation, proteolytic processing, cofactor stimulation and inhibition, each of which represents a possible point of therapeutic intervention. Recent research suggests that, in addition to the direct inhibition of ADAMTS proteinases with low molecular weight non-peptidic inhibitors, targeting the transcription and protein processing of these enzymes could be effective therapeutic approaches.

Molecular events in tendinopathy: a role for metalloproteases.

Disorganized, haphazard ineffective healing is a constant feature of chronic tendinopathy. Normal tendon is composed mostly of type I collagen. Tendinopathic tendons, conversely, have a greater proportion of type III collagen, which is associated with tendon rupture. Matrix metalloproteinases (MMPs) are involved in remodelling of the extracellular matrix (ECM) of tendons, because they are either up- or down-regulated in tendinopathy. A balance between MMPs and tissue inhibitors of metalloproteinases is required to maintain tendon homeostasis. The mechanism of activation of MMPs is poorly understood, and their precise role in tendinopathy is unclear.