Matrix Enzyme Research Articles

Mechanical loading of cartilage explants with joint-specific loading patterns modulates gene expression of lubricin and catabolic matrix enzymes

• Excessive mechanical loading can lead to damage and loss of matrix components due to the synthesis of catabolic enzymes such as MMPs and ADAMTS. • Cartilaginous surfaces, such as the temporomandibular joint (TMJ), spend most of the time in relative motion with a constant translation of the contact zone [1,2]. • Such migrating contacts are essential for tissues function [3]. • Recent mechanobiological studies almost exclusively apply relatively simple loading regimens such as uniaxial compressive and/or shear forces while keeping the contact area stationary. 

From the ECM to Thrombosis: a New Role for the Matrix Enzyme Lysyl Oxidase

IntroductionLysyl oxidase (LOX) catalyzes the oxidation of lysines to aldehydes, providing stability in connective tissues through spontaneous cross‐linking of collagen and elastin. LOX expression is elevated in sera of patients with myeloproliferative neoplasms (MPN), a group of disorders that have in common the proliferation of megakaryocytes and myelofibrosis in bone marrow. Thrombosis is a leading cause of morbidity and mortality in MPN, yet there is still controversy over contributors to the thrombotic events in MPN.Objectiveto test whether Lysyl Oxidase (LOX) is involved in the pathogenesis of thrombosis.MethodsA transgenic mouse model expressing LOX in platelets (Pf4‐Loxtg/tg) was used to analyze platelet activation and thrombus formation.ResultsVessel occlusion after endothelial injury was significantly shorter in Pf4‐Loxtg/tg mice, indicating a higher propensity for thrombus formation in vivo. While exploring mechanisms involved, we found that Pf4‐Loxtg/tg platelets adhere better to collagen. The higher affinity of Pf4‐Loxtg/tg platelets to monomeric compared to fibrillar collagen, as well as affinity to the GFOGER peptide implies integrin α2β1 as the collagen receptor targeted by LOX. Taken together, our study identified LOX as a new regulator of collagen receptor and thus platelet activation and thrombosis.

Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement.

ObjectiveShort-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal-recessive ECHS1 deficiency.MethodsUsing exome sequencing, we identified ten unrelated individuals carrying compound heterozygous or homozygous mutations in ECHS1. Functional investigations in patient-derived fibroblast cell lines included immunoblotting, enzyme activity measurement, and a palmitate loading assay.ResultsPatients showed a heterogeneous phenotype with disease onset in the first year of life and course ranging from neonatal death to survival into adulthood. The most prominent clinical features were encephalopathy (10/10), deafness (9/9), epilepsy (6/9), optic atrophy (6/10), and cardiomyopathy (4/10). Serum lactate was elevated and brain magnetic resonance imaging showed white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism. Analysis of patients’ fibroblast cell lines (6/10) provided further evidence for the pathogenicity of the respective mutations by showing reduced ECHS1 protein levels and reduced 2-enoyl-CoA hydratase activity. While serum acylcarnitine profiles were largely normal, in vitro palmitate loading of patient fibroblasts revealed increased butyrylcarnitine, unmasking the functional defect in mitochondrial β-oxidation of short-chain fatty acids. Urinary excretion of 2-methyl-2,3-dihydroxybutyrate – a potential derivative of acryloyl-CoA in the valine catabolic pathway – was significantly increased, indicating impaired valine oxidation.InterpretationIn conclusion, we define the phenotypic spectrum of a new syndrome caused by ECHS1 deficiency. We speculate that both the β-oxidation defect and the block in l-valine metabolism, with accumulation of toxic methacrylyl-CoA and acryloyl-CoA, contribute to the disorder that may be amenable to metabolic treatment approaches.

Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-β hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-β hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

Whole Exome Sequencing Reveals DYSF, FKTN, and ISPD Mutations in Congenital Muscular Dystrophy Without Brain or Eye Involvement.

BackgroundCongenital muscular dystrophies (CMDs) are a genetically and clinically heterogeneous group of neuromuscular disorders. Several genes encoding extracellular matrix, nuclear envelope, sarcolemmal proteins and glycosylation enzymes have been implicated in CMDs. The large overlap of clinical presentations due to mutations in different genes poses a challenge for clinicians in determining disease etiology for each patient.ObjectiveWe investigated the use of whole exome sequencing (WES) in identifying the genetic cause of disease in 5 CMD patients from 3 families who presented with highly similar clinical features, including early-onset rapidly progressive weakness without brain or eye abnormalities.MethodsWhole exome sequencing was performed on DNA from affected individuals. Potential functional impacts of mutations were investigated by immunostaining on available muscle biopsies.ResultsPathogenic mutations in 3 different genes, DYSF, FKTN, and ISPD were identified in each family. Mutation in DYSF led to absence of dysferlin protein in patient muscle. Mutations in ISPD led to impaired ISDP function, as demonstrated by deficiency of α-dystroglycan glycosylation in patient muscle.ConclusionsThis study highlights the benefit of unbiased genomic approaches in molecular diagnosis of neuromuscular disorders with high clinical heterogeneity, such as the phenotypes observed in our patients. Our results suggest that dysferlin deficiency should be in the differential diagnosis of congenital and rapidly progressive muscular dystrophy, and therefore dysferlin antibody should be in the standard immunohistochemistry panel for muscle biopsies in cases with suspected CMD.

Effects of Bisphosphonate on the Expression of Matrix Enzymes during Endochondral Ossification

Bisphosphonates have been reported to have chondroprotective activities in addition to its original functions. However, mechanisms for these just began to be elucidated. Under the hypothesis that bisphosphonates may regulate expression and activities of matrix enzymes during degradation of cartilage for bone formation, we administrated an alendronate (1 mg/kg) to newborn rats subcutaneously once a day for 4, 7, and 10 days. To identify the effects of alendronate on cartilage, thickness of cartilage layer was measured by histomorphometry on the proximal epiphysis of tibia. Immunofluorescence staining and RT-PCR were performed to investigate the expressions of matrix enzymes in both in vitro and in vivo. MTS assay revealed that at 10 -3 M in concentration, alendronate significantly reduced viability of chondrocytes. The mRNA expressions of MMP-1, MMP-9, EMMPRIN, and TIMP-3 in primary chondrocytes were decreased by the alendronate treatment. Interestingly, TIMP-1 mRNA expression was significantly increased, whereas a constitutive form, TIMP-2 was relatively unchanged by the treatment. The thickness of proliferating layer at postnatal day 7 was not significantly different, whereas thickness of hypertrophied layer was significantly thicker in the alendronate group than in the control (p These results suggest that the alendronate have chondroprotective properties by down-regulation of MMPs and up-regulation of TIMPs during endochondral ossification.

Phenotypic Variability and Newly Identified Mutations of the <i>IVD </i>Gene in Japanese Patients with Isovaleric Acidemia

Isovaleric acidemia (IVA) is an autosomal recessive inborn error affecting leucine metabolism. It is caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD), a mitochondrial matrix enzyme that catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. IVD is a FAD-containing enzyme, consisting of four identical subunits. Clinical features of IVA include poor feeding, vomiting, lethargy, developmental delay, metabolic acidosis, and a characteristic "sweaty foot" odor. IVA is one of the target disorders for newborn screening by tandem mass spectrometry (MS/MS). The human IVD gene is located on chromosome 15q. To date, over 50 disease-causing mutations have been reported worldwide. In this study, we searched for IVD mutations in five Japanese patients with IVA (neonatal type, two patients; chronic intermittent type, two patients; and mild biochemical type, one patient). The diagnosis of IVA was confirmed by urinary organic acid analysis using gas chromatography and mass spectrometry. All coding exons and the flanking introns in the IVD gene were amplified by PCR and were directly sequenced. We thus identified six hitherto unknown mutations (p.G94D, p.E116K, p.M167T, p.L243P, p.L246P, and c.696+1G>T) and four previously reported (p.R53P, p.R395C, p.Y403C, and p.E411K) pathogenic mutations. All patients were compound heterozygotes, and each mutation was identified in a single patient. Pathogenicity of newly identified mutations was validated using computational programs. Among them, the p.M167T is believed to influence FAD binding, as the position 167 is present in one of the FAD-binding sites. Our results have illustrated the heterogeneous mutation spectrum and clinical presentation of IVA in the Japanese patients.

Combined inhibition of tumor necrosis factor α and interleukin-17 as a therapeutic opportunity in rheumatoid arthritis: development and characterization of a novel bispecific antibody.

Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo. Cultures of human fibroblast-like synoviocytes (FLS) were stimulated with TNFα, IL-17, or a combination of both. Single/combined neutralizing antibodies against TNFα and IL-17 were used to examine in vitro cytokine responses and in vivo development of arthritis and bone and cartilage destruction in TNFα-transgenic mice. Bispecific anti-TNFα/IL-17 antibodies were designed, and their potential to block cytokine responses in human FLS was tested. TNFα and IL-17 had additive/synergistic effects in promoting production of IL-6, IL-8, and granulocyte colony-stimulating factor, as well as matrix metalloproteinases, in FLS. Bispecific anti-TNFα/IL-17 antibodies showed superior efficacy in blocking cytokine and chemokine responses in vitro. Furthermore, dual versus single inhibition of both cytokines using neutralizing antibodies was more effective in inhibiting the development of inflammation and bone and cartilage destruction in arthritic mice. Combined blockade of TNFα and IL-17 was more effective than single blockade in inhibiting cytokine, chemokine, and matrix enzyme responses from human mesenchymal cells and in blocking tissue destruction associated with arthritis, and additionally showed a positive impact on rebalance of bone homeostasis. Bispecific anti-TNFα/IL-17 antibodies may have superior efficacy in the treatment of arthritis and may overcome the limited therapeutic responses obtained with single cytokine neutralization.

A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila.

The apical extracellular matrix plays a central role in epithelial tube morphogenesis. In the Drosophila tracheal system, Serpentine (Serp), a secreted chitin deacetylase expressed by the tracheal cells plays a key role in regulating tube length. Here, we show that the fly fat body, which is functionally equivalent to the mammalian liver, also contributes to tracheal morphogenesis. Serp was expressed by the fat body, and the secreted Serp was taken up by the tracheal cells and translocated to the lumen to functionally support normal tracheal development. This process was defective in rab9 and shrub/vps32 mutants and in wild-type embryos treated with a secretory pathway inhibitor, leading to an abundant accumulation of Serp in the fat body. We demonstrated that fat body-derived Serp reached the tracheal lumen after establishment of epithelial barrier function and was retained in the lumen in a chitin synthase-dependent manner. Our results thus reveal that the fat body, a mesodermal organ, actively contributes to tracheal development.

Biocatalytic polymer nanofibers for stabilization and delivery of enzymes

Electrospun water-soluble nanofibers as enzyme supports have potential to overcome limitations of traditional immobilization by increasing the surface area-to-volume ratio, reducing diffusional limitations, and improving storage stability. In this study, β-galactosidase (lactase, Aspergillus oryzae) was electrospun into polyethylene oxide (PEO) nanofibers with Pluronic F-127 (F127) (polyethylene oxide/polypropylene oxide block copolymer) to enable dry storage stability and subsequent rapid dissolution upon use in applications such as sensing, bioactive packaging, and bioprocessing. Nanofibers were cylindrical (average diameter ∼300nm) and exhibited no beading after spinning or storage. After electrospinning, enzymes retained up to 92% of their native activity. After 4 weeks storage, the immobilized enzyme fiber retained up to 44% of the activity of the original fiber. These results suggest that co-spinning enzymes in a polymer nanofiber matrix can improve activity retention and dry storage stability compared to traditional immobilization methods and enables rapid dissolution for ease of use.

Ocena aktywności wybranych enzymów macierzy pozakomórkowej w przebiegu gojenia doświadczalnych uszkodzeń termicznych

Dobrze poznana sekwencja zmian ekspresji metaloproteinaz macierzy (ang. matrix metalloproteinases – MMPs) w przebiegu poszczególnych faz naprawy uszkodzeń tkankowych pozwoliła Autorom pracy na zastosowanie doświadczalnego modelu gojenia oparzeń, który umożliwił porównanie terapeutycznej aktywności P

618PD - A Phase 2 Randomized, Double-Blind, Placebo Controlled Study of Simtuzumab or Placebo in Combination with Gemcitabine for the First Line Treatment of Pancreatic Adenocarcinoma

ABSTRACT Aim: Simtuzumab (SIM) is a humanized antibody that inhibits lysyl oxidase-like molecule 2 (LOXL2), an extracellular matrix enzyme that catalyzes the covalent cross-linking of collagen and is widely expressed across desmoplastic tumors. Inhibiting LOXL2 is expected to block formation of desmoplasia, which is thought to play an important role in tumor progression and metastasis. In the Phase 1 study, SIM was safe and well-tolerated in patients (pts) with advanced solid tumors and showed early evidence of efficacy. Based on these results, a randomized, double-blind, placebo-controlled phase 2 study of SIM + gemcitabine (Gem) vs. placebo (pbo) + Gem in pts with metastatic pancreatic adenocarcinoma was initiated. Methods: Eligible pts had no prior systemic therapy for advanced disease and Eastern Cooperative Oncology Group performance status of 0 or 1. The primary endpoint was progression free survival (PFS). Pts were randomized to 200 mg SIM, 700 mg SIM, or pbo at a 1:1:1 ratio in combination with Gem in cycles of 28 days. In each cycle, pts received IV SIM or pbo infused on Days 1 and 15, and IV Gem (1000 mg/m2) on Days 1, 8, and 15. Results: Between 3/28/12 and 5/15/13, 236 pts were randomized and treated; 76 pts (200 mg SIM/Gem), 79 pts (700 mg SIM/Gem), and 81 pts (pbo/Gem). Median PFS was 3.5 (HR 1.12; p = 0.63 vs pbo), 3.7 (HR 1.08; p = 0.75 vs pbo), and 3.7 months for 200 mg SIM/Gem, 700 mg SIM/Gem, and pbo/Gem arms, respectively. Median overall survival (OS) was 5.9 (HR 1.05; p = 0.76 vs pbo), 7.6 (HR 0.83; p = 0.26 vs pbo), and 5.7 months, respectively. Gemcitabine-related expected toxicities included anemia, thrombocytopenia, neutropenia, and nausea. There were no differences in the safety profile of the SIM plus Gem groups versus pbo plus Gem group. Conclusions: The addition of SIM to Gem does not improve PFS or OS in advanced pancreatic cancer pts. Disclosure: H. Dong: is an employee of Gilead Sciences, the sponsor of the clinical study, and holds shares of Gilead stock; D. Thai: is an employee of Gilead Sciences, the sponsor of the clinical study, and holds shares of Gilead stock. All other authors have declared no conflicts of interest.

Effect of osteopontin on TIMP-1 and TIMP-2 mRNA in chondrocytes of human knee osteoarthritis in vitro.

Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of matrix metalloproteinases (MMPs) and enzymes from the a disintegrin and metalloproteinase domain with thrombospondin motifs family in osteoarthritis (OA). Elevated osteopontin (OPN) levels in plasma, synovial fluid and articular cartilage are associated with progressive OA joint damage; however, the role of OPN in the pathological changes of knee OA remains undetermined. The present study was undertaken to examine the effect of OPN on the expression of TIMP-1 and TIMP-2 mRNA in chondrocytes from 16 patients with knee OA. In this study, following the stimulation of human chondrocytes with recombinant human OPN (rhOPN; 100 ng/ml and 1 μg/ml, respectively) for 48 h, MTT assay was used to determine cell viability while the quantitative polymerase chain reaction (PCR) was used to detect the alterations in TIMP-1 and TIMP-2 levels. The results illustrated that neither 100 ng/ml nor 1 μg/ml rhOPN caused cytotoxicity or apoptosis of chondrocytes and that the relative mRNA expression of TIMP-1 and TIMP-2 was significantly increased in the 1 μg/ml rhOPN group compared with that in the control group (P=0.022 and P=0.003, respectively). However, no significant difference in expression was revealed between the 100 ng/ml rhOPN and control groups (P=0.998 and P=0.209, respectively). In conclusion, OPN may have a protective effect against pathological changes in advanced-stage OA.

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues

Mammalian mitochondria are able to produce formate from one-carbon donors such as serine, glycine, and sarcosine. This pathway relies on the mitochondrial pool of tetrahydrofolate (THF) and several folate-interconverting enzymes in the mitochondrial matrix. We recently identified MTHFD2L as the enzyme that catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. We show here that the MTHFD2L enzyme is bifunctional, possessing both CH2-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities. The dehydrogenase activity can use either NAD(+) or NADP(+) but requires both phosphate and Mg(2+) when using NAD(+). The NADP(+)-dependent dehydrogenase activity is inhibited by inorganic phosphate. MTHFD2L uses the mono- and polyglutamylated forms of CH2-THF with similar catalytic efficiencies. Expression of the MTHFD2L transcript is low in early mouse embryos but begins to increase at embryonic day 10.5 and remains elevated through birth. In adults, MTHFD2L is expressed in all tissues examined, with the highest levels observed in brain and lung.

Are There Protective Genes for Disc Degeneration?

Intervertebral disc degeneration is a major cause of back pain with a huge impact to the quality of life as we age. It is clear from studies that there is a significant genetic contribution to the susceptibility of an individual to disc degeneration. Indeed, several genetic risk factors have been identified that included extracellular matrix proteins and enzymes that modify these proteins. These genetic risk factors interact with the environment that impact on the onset, severity, and symptomatic outcomes. In a population study in Hong Kong, whereas over 90% of most people will succumb to some form of disc degeneration by the age of 50 years, there are individuals who showed no indication of disc degeneration by MRI suggesting the possibility of “protective factors,” either genetic and/or environmental.1 It is likely that genetics would play a role in protective mechanisms. It is possible to study potential protective genes in human using case-control association studies, but it would require the collection of a very large cohort of “protected” individuals. Another approach is to study in animal models and mapped the identified loci to the human genome. In mice, there are “super healer” strains with superior healing potentials of damaged tissues including elastic cartilage of the ear and hyaline cartilage of synovial joints.2 Whether these healing potentials translate to protective mechanisms of the intervertebral disc is not clear and has not been studied. Our initial analysis comparing good healer (Lg/J and MRL) with poor healer (Sm/J and C57B) strains of mice suggests that there should be protective genes influencing the homeostasis of the nucleus pulposus and the annulus fibrosus. A gene discovery study has been performed for knee joint cartilage repair in these mice and potential genetic loci for improved cartilage repair identified.3,4 Thus, with the establishment of appropriate phenotypic traits for the intervertebral disc, it is possible to search for protective/repair genes for the disc. There should be “protective genes”; what is important is finding the appropriate animal models and human cohorts for genetic studies and validation. Focusing on protective genes and understanding the molecular and cellular controls involved in delaying the onset and progression of intervertebral disc degeneration may serve to be a better approach in developing therapeutic treatments and preventative strategies. Disclosure of Interest None declared References Jim JJ, Noponen-Hietala N, Cheung KM, et al. The TRP2 allele of COL9A2 is an age-dependent risk factor for the development and severity of intervertebral disc degeneration. Spine 2005;30(24):2735–2742 Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS, Little CB. Evidence for articular cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage 2008;16(11):1319–1326 Rai MF, Hashimoto S, Johnson EE, et al. Heritability of articular cartilage regeneration and its association with ear wound healing in mice. Arthritis Rheum 2012;64(7):2300–2310 Rai MF, Schmidt EJ, McAlinden A, Cheverud JM, Sandell LJ. Molecular insight into the association between cartilage regeneration and ear wound healing in genetic mouse models: targeting new genes in regeneration. G3 (Bethesda) 2013;3(11):1881–1891

Glycosomal Targets for Anti-Trypanosomatid Drug Discovery

Glycosomes are peroxisome-related organelles found in all kinetoplastid protists, including the human pathogenic species of the family Trypanosomatidae: Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. Glycosomes are unique in containing the majority of the glycolytic/gluconeogenic enzymes, but they also possess enzymes of several other important catabolic and anabolic pathways. The different metabolic processes are connected by shared cofactors and some metabolic intermediates, and their relative importance differs between the parasites or their distinct lifecycle stages, dependent on the environmental conditions encountered. By genetic or chemical means, a variety of glycosomal enzymes participating in different processes have been validated as drug targets. For several of these enzymes, as well as others that are likely crucial for proliferation, viability or virulence of the parasites, inhibitors have been obtained by different approaches such as compound libraries screening or design and synthesis. The efficacy and selectivity of some initially obtained inhibitors of parasite enzymes were further optimized by structure-activity relationship analysis, using available protein crystal structures. Several of the inhibitors cause growth inhibition of the clinically relevant stages of one or more parasitic trypanosomatid species and in some cases exert therapeutic effects in infected animals. The integrity of glycosomes and proper compartmentalization of at least several matrix enzymes is also crucial for the viability of the parasites. Therefore, proteins involved in the assembly of the organelles and transmembrane passage of substrates and products of glycosomal metabolism offer also promise as drug targets. Natural products with trypanocidal activity by affecting glycosomal integrity have been reported.

Obesity and lipid stress inhibit carnitine acetyltransferase activity

Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

Inhibition of TAK1 and/or JAK Can Rescue Impaired Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Osteoarthritis-Like Conditions

To rescue chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in osteoarthritic conditions by inhibition of protein kinases. hMSCs were cultured in pellets. During early chondrogenic differentiation, these were exposed to osteoarthritic synovium-conditioned medium (OAS-CM), combined with the Janus kinase (JAK)-inhibitor tofacitinib and/or the transforming growth factor β-activated kinase 1 (TAK1)-inhibitor oxozeaenol. To evaluate effects on chondrogenesis, the glycosaminoglycan (GAG) content of the pellets was measured at the time that chondrogenesis was manifest in control cultures. Moreover, mRNA levels of matrix molecules and enzymes were measured during this process, using real-time polymerase chain reaction (RT-PCR). Initial experiments were performed with hMSCs from a fetal donor, and results of these studies were confirmed with hMSCs from adult donors. Exposure to OAS-CM resulted in pellets with a much lower GAG content, reflecting inhibited chondrogenic differentiation. This was accompanied by decreased mRNA levels of aggrecan, type II collagen, and Sox9, and increased levels of matrix metalloproteinase (MMP)1, MMP3, MMP13, ADAMTS4, and ADAMTS5. Both tofacitinib (JAK-inhibitor) and oxozeaenol (TAK1 inhibitor) significantly increased the GAG content of the pellets in osteoarthritis (OA)-like conditions. The combination of both protein kinase inhibitors showed an additive effect on GAG content. In agreement with this, in the presence of OAS-CM, both tofacitinib and oxozeaenol increased mRNA expression of sox9. The expression of aggrecan and type II collagen was also up-regulated, but this only reached significance for aggrecan after TAK1 inhibition. Both inhibitors decreased the mRNA levels of MMP1, 3, and 13 in the presence of OAS-CM. Moreover, oxozeaenol also significantly down-regulated the mRNA levels of aggrecanases ADAMTS4 and ADAMTS5. When combined, the inhibitors caused additive reduction of OA-induced MMP1 mRNA expression. Counteraction of OAS-CM-induced inhibition of chondrogenesis by these protein kinase inhibitors was confirmed with hMSCs of two different adult donors. Both tofacitinib and oxozeaenol significantly improved GAG content in cell pellets from these adult donors. Tofacitinib and oxozeaenol partially prevent the inhibition of chondrogenesis by factors secreted by OA synovium. Their effects are additive. This indicates that these protein kinase inhibitors can potentially be used to improve cartilage formation under the conditions occurring in osteoathritic, or otherwise inflamed, joints.

Brazilin blocks the induction of NFKB1/P50 in human chondrocytes and synoviocytes

Purpose: Brazilin is one major constituent of Caesalpinia sappan and is known to possess anti-bacterial, anti-oxidative, and anti-inflammatory activities. Osteoarthritis (OA) creates a disequilibrium between the catabolic and anabolic activities of chondrocytes in remodeling the cartilage extracellular matrix (ECM). Pro-inflammatory signaling contributes to the degradation of the cartilage matrix. Under OA conditions, synoviocytes produce a range of inflammatory mediators, including IL-1β. Upon binding to chondrocytes, these mediators activate specific signal transduction cascades, such as the nuclear factor-kappa-B (NF-κB) pathway, which acts as one central regulator of the catabolic processes that suppresses the synthesis of ECM components, and induces the expression of matrix metalloproteinases and enzymes of the ADAMTS family, leading to perpetuated cartilage breakdown. This study aimed to evaluate the effects of brazilin in osteoarthritic chondrocytes and synoviocytes with particular focus on the NF-κB pathway. Methods: Brazilin was isolated from Caesalpinia sappan extract (CSE) and identified using HPLC and NMR methods. Chondrocytes and synoviocytes were isolated from OA patients undergoing total knee replacement surgery. Cells were either left untreated for control samples, were treated with 10 ng/ml IL-1β, or were pre-treated with 10 μg/ml brazilin prior to the addition of 10 ng/ml IL-1β. Genes associated with NF-κB-mediated signal transduction were examined in primary human chondrocytes using an NF-κB signaling array and in silico pathway analysis. The induction and inhibition of NFKB1, its gene product p50, and the precursor p105 were investigated in primary human chondrocytes and synoviocytes using RT-qPCR and Western blotting. Results: Nine NF-κB pathway-related genes (BIRC3, CCL2, CSF1, CSF3, IL1B, IL8, NFKB1, NFKB2 and TNFAIP3) were found to be significantly up-regulated by IL-1β-stimulation and significantly down-regulated after pre-incubation with brazilin. Pathway analysis revealed NFKB1 as one major gene regulating the anti-inflammatory activities of brazilin. RT-qPCR assays confirmed that the up-regulation of NFKB1 mRNA in IL-1β-stimulated primary chondrocytes and synoviocytes was significantly reduced by pre-treatment with brazilin. Western blotting showed that IL-1β treatment increased p105 protein in chondrocytes with a peak after 1 h followed by a steady decline, while increasing constantly the amount of p50 over 24 h. Brazilin suppressed the IL-1β-mediated induction of NFKB1/p50 in chondrocytes and synoviocytes. Conclusions: The present study suggests that brazilin effectively blocks the induction of NFKB1/p50 in cytokine-stimulated primary human chondrocytes and synoviocytes, pointing towards a chondroprotective potential of brazilin, which may be beneficial for reducing cartilage breakdown in OA.

A Novel SUCLA2 Mutation in a Portuguese Child Associated With “Mild” Methylmalonic Aciduria

Succinyl-coenzyme A synthase is a mitochondrial matrix enzyme that catalyzes the reversible synthesis of succinate and adenosine triphosphate (ATP) from succinyl-coenzyme A and adenosine diphosphate (ADP) in the tricarboxylic acid cycle. This enzyme is made up of α and β subunits encoded by SUCLG1 and SUCLA2, respectively. We present a child with severe muscular hypotonia, dystonia, failure to thrive, sensorineural deafness, and dysmorphism. Metabolic investigations disclosed hyperlactacidemia, moderate urinary excretion of methylmalonic acid, and elevated levels of C4-dicarboxylic carnitine in blood. We identified a novel homozygous p.M329V in SUCLA2. In cultured cells, the p.M329V resulted in a reduced amount of the SUCLA2 protein, impaired production of mitochondrial ATP, and enhanced production of reactive oxygen species, which was partially reduced by using 5-aminoimidazole-4-carboxamide ribonucleotide in the culture medium. Expanding the array of SUCLA2 mutations, we suggested that reactive oxygen species scavengers are likely to impact on disease prognosis.