Transcriptional induction of ADAMTS5 protein by nuclear factor-κB (NF-κB) family member RelA/p65 in chondrocytes during osteoarthritis development.

Here we sought to identify transcription factors that induce ADAMTS5, a crucial proteinase for osteoarthritis development. Exhaustive comparison of the genomic sequences of human, macaque, and mouse ADAMTS5 genes revealed that the proximal 1.4 kb of the 5'-end-flanking regions containing several consensus motifs was highly conserved. Among putative transcription factors for these motifs, NF-κB family member RelA/p65 most strongly stimulated the promoter activity. In the ADAMTS5 gene, there were three NF-κB binding motifs, in which deletion, mutagenesis, and tandem repeat analyses of the luciferase assay identified the core responsive elements of RelA/p65 to be -896/-887 and -424/-415 bp with specific bindings. The endogenous Adamts5 expression in ATDC5 cells was increased by RelA/p65 overexpression and decreased by knockdown through its siRNA. The expression was also inhibited by the Rela deletion through Cre transfection in primary articular chondrocytes from Rela(fl/fl) mice. In the ex vivo culture of femoral head cartilage from mesenchymal cell-specific Rela knock-out (Prx1-Cre;Rela(fl/fl)) mice, aggrecanolysis was significantly lower than that in the Rela(fl/fl) cartilage. Finally, in the experimental mouse osteoarthritis model, ADAMTS5 and RelA were co-localized in chondrocytes of degraded articular cartilage. We conclude that RelA/p65 is a potent transcriptional activator of ADAMTS5 in chondrocytes during osteoarthritis development.

GSK-3α and GSK-3ß proteins are involved in early stages of chondrocyte differentiation with functional redundancy through RelA protein phosphorylation.

Here we examine the roles of two isoforms of glycogen synthase kinase-3 (GSK-3), GSK-3α and GSK-3ß, in skeletal development. Both isoforms were unphosphorylated and active in chondrocyte differentiation stages during SOX9 and type II collagen (COL2A1) expression. Although knock-out of both alleles of Gsk3a (Gsk3a(-/-)) or a single allele of Gsk3b (Gsk3b(+/-)) in mice did not significantly affect skeletal development, compound knock-out (Gsk3a(-/-);Gsk3b(+/-)) caused dwarfism with impairment of chondrocyte differentiation. GSK-3α and GSK-3ß induced differentiation of cultured chondrocytes with functional redundancy in a cell-autonomous fashion, independently of the Wnt/ß-catenin signal. Computational predictions followed by SOX9 and COL2A1 transcriptional assays identified RelA (NF-κB p65) as a key phosphorylation target of GSK-3. Among several phosphorylation residues in RelA, Thr-254 was identified as the critical phosphorylation site for GSK-3 that modulated chondrocyte differentiation. In conclusion, redundant functions of GSK-3α and GSK-3ß through phosphorylation of RelA at Thr-254 play a crucial role in early stages of chondrocyte differentiation.

Early functional improvement after a modified ligament reconstruction tendon interposition arthroplasty for thumb basal joint arthritis.

Many modifications to trapeziectomy have been proposed for the treatment of thumb basal joint arthritis. Their final outcomes have been discussed intensively, whereas functional changes in the early post-operative period have not been fully documented. The purpose of the present study is to evaluate an early functional change following our modified ligament reconstruction with tendon interposition (LRTI) arthroplasty. Nine patients (ten thumbs) were included in this study. Pain levels, strength, and mobility were assessed before and after surgery at intervals of two months. Pain level significantly improved at two months after surgery. Grip and pinch strengths had increased gradually after a temporal decrease at two-month follow-up, and were significantly stronger at six months after surgery. Palmar abduction improved significantly at six months after surgery, whereas opposition did not change significantly. A modified LRTI is an effective procedure in terms of early functional improvement of pain, strength, and mobility.

[Pre-analytical problems on assay conditions for blood midkine].

We have established the assay conditions of midkine (MK) measurement, the reference intervals and evaluation for clinical significance of blood MK measurement. MK is a kind of cytokines and basic protein which is a heparin-binding growth factor of various cells. The increase of MK expression suggests a prognostic value in early stage on cancers or inflammation. But significant problems in the MK measurement are alterations resulting from standing time and temperature instability after blood collection. Assay of MK was performed with solid phase human MK immunoassay recently developed sensitive enzyme linked immunosorbent method. The assay condition of MK was required to be separated immediately after blood sampling within 24 hrs at 4 degrees C or within 2 hrs at room temperature-standing. Plasma sample obtained with EDTA-2Na or citric acid-Na, and serum obtained from plain tube container showed good results. Linearity was obtained up to 1500 pg/ml and repeatability and reproducibility were within 10% as CV%. The recovery of MK was 101.1+/-3.8% with 10 specimens ranged 97-105%. Addition of interfering substances showed no effect on assay results when hemoglobin, EDTA-Na, citrate and turbidity check, but conjugated bilirubin (over 0.68 mmol/l) and gave negative errors within 10% in the assay results and heparin gave negative errors. The reference interval was 550 +/- 160 pg/ml in healthy individuals serum.

Biphasic regulation of chondrocytes by Rela through induction of anti-apoptotic and catabolic target genes.

In vitro studies have shown that Rela/p65, a key subunit mediating NF-κB signalling, is involved in chondrogenic differentiation, cell survival and catabolic enzyme production. Here, we analyse in vivo functions of Rela in embryonic limbs and adult articular cartilage, and find that Rela protects chondrocytes from apoptosis through induction of anti-apoptotic genes including Pik3r1. During skeletal development, homozygous knockout of Rela leads to impaired growth through enhanced chondrocyte apoptosis, whereas heterozygous knockout of Rela does not alter growth. In articular cartilage, homozygous knockout of Rela at 7 weeks leads to marked acceleration of osteoarthritis through enhanced chondrocyte apoptosis, whereas heterozygous knockout of Rela results in suppression of osteoarthritis development through inhibition of catabolic gene expression. Haploinsufficiency or a low dose of an IKK inhibitor suppresses catabolic gene expression, but does not alter anti-apoptotic gene expression. The biphasic regulation of chondrocytes by Rela contributes to understanding the pathophysiology of osteoarthritis.

Reproducibility of measurements of thumb abduction.

The reproducibilities of various measurements of thumb abduction were compared. Two independent observers measured the thumb abduction in 30 volunteers by the following four methods: distance between the thumb tip and the flexion crease of the index finger proximal interphalangeal joint; distance between the flexion crease of the thumb interphalangeal joint and the proximal palmar crease; angle between the thumb and index metacarpals; and angle between the thumb and index proximal phalanx. Measurements were repeated in three weeks and their reproducibility was assessed by the intraclass correlation coefficient (ICC). Intra-observer reproducibilities by distances were high with ICCs between 0.74 and 0.89, compared with those by angles with ICCs between 0.28 and 0.71. Inter-observer reproducibilities by distances were also high (ICCs = 0.79 and 0.81) compared with those by angles (ICCs = 0.28 and 0.42). Assessment of thumb abduction based on distance in the first web is recommended in terms of reproducibility.