Comparison of splinting immobilization and K-wire fixation in children with type II phalange neck fracture.

To compare outcomes of type II phalangeal neck fractures in children who received closed reduction followed by splinting immobilization or by K-wire fixation. Furthermore, we analyzed the remodeling potential of residual deformities and the relationship between age and outcomes. Patients in Children's Hospital of Fudan University, Xiamen Hospital were included in the study from October 2015 to October 2018. We compared outcomes between the conservation group and operation group. Remodeling of residual deformities was calculated on a series of anteroposterior and lateral radiography. The correlation between age and outcomes was analyzed using Spearman's rank correlation coefficient. Forty patients (25 males) were enrolled. Nineteen patients had subtype IIa, 19 subtype IIb, and two subtype IIc fractures. Left hands were affected more than right hands, and small finger and proximal phalanx were more frequently involved. There were no significant differences between conservation group and operation group among excellent, good, and fair outcomes. And the outcomes were not significantly different between the IIa and IIb subtypes. An average sagittal remodeling rate was 88.5%, and coronal remodeling rate was 56.71%, respectively, in 13 patients with residual deformities. There was a significant correlation between age and final outcomes. Closed reduction and stable splint fixation may be an effective and economical initial treatment option. Fracture subtype does not seem to be a key factor for choosing treatment options. The fractured phalangeal neck had remodeling potential whether on sagittal or coronal plane. Younger age might be a predictor of better outcomes in children with type II phalanx neck fractures.

E74-like factor 3 (ELF3) impacts on matrix metalloproteinase 13 (MMP13) transcriptional control in articular chondrocytes under proinflammatory stress.

Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position -78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1ß stimulation in chondrocytes, and the IL-1ß-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3(-/-) mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1ß-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1beta (IL-1beta). We reported previously that early activation of EGR-1 by IL-1beta results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-kappaB-dependent inhibition by IL-1beta. Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1beta, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1beta. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1beta colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at -403/-381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.

Responses to the proinflammatory cytokines interleukin-1 and tumor necrosis factor alpha in cells derived from rheumatoid synovium and other joint tissues involve nuclear factor kappaB-mediated induction of the Ets transcription factor ESE-1.

OBJECTIVE: To investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor kappaB (NF-kappaB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines. METHODS: ESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase-polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor kappaB (IkappaB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-kappaB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-kappaB family were conducted using the NF-kappaB site from the ESE-1 promoter as a probe. RESULTS: Immunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1beta and TNFalpha in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1beta requires activation of NF-kappaB and binding of p50 and p65 family members to the NF-kappaB site in the ESE-1 promoter. Overexpression of IkappaB using an adenoviral vector blocked IL-1beta-induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-kappaB binds to the ESE-1 promoter in vivo. CONCLUSION: ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1beta, TNFalpha, or LPS. This induction relies on the translocation of the NF-kappaB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-kappaB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.

Egr-1 mediates transcriptional repression of COL2A1 promoter activity by interleukin-1beta.

Following induction and activation of the early growth response (Egr)-1 transcription factor in human chondrocytes, interleukin-1beta (IL-1beta) suppresses the expression of the type II collagen gene (COL2A1), associated with induction of Egr-1 binding activity in nuclear extracts. The COL2A1 proximal promoter contains overlapping binding sites for Egr-1 and Sp1 family members at -119/-112 bp and -81/-74 bp. Mutations that block binding of Sp1 and Sp3 to either site markedly reduce constitutive expression of the core promoter. IL-1beta-induced Egr-1 binds strongly to the -119/-112 bp site, and mutations that block Egr-1 binding prevent inhibition by IL-1beta. Cotransfection with pCMV-Egr1 potentiates the inhibition of COL2A1 promoter activity by IL-1beta, whereas overexpression of dominant-negative Egr-1 mutant, Wilm's tumor-1 (WT1)/Egr1, Sp1, or Sp3 reverses the inhibition by IL-1beta. Cotransfection of pGL2-COL2/Gal4, in which we substituted the critical residue for Egr-1 binding with a Gal4 binding domain and a pCMV-Gal4-Egr1 chimera permits an inhibitory response to IL-1beta that is reversed by overexpression of Gal4-CBP. Our results indicate that IL-1beta-induced activation of Egr-1 binding is required for inhibition of COL2A1 proximal promoter activity and suggest that Egr-1 acts as a repressor of a constitutively expressed collagen gene by preventing interactions between Sp1 and the general transcriptional machinery.

The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-gamma-mediated inhibition in human chondrocytes: requirement for Stat1 alpha, Jak1 and Jak2.

Interferon-gamma (IFN-gamma) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene ( COL2A1 ) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-gamma and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-gamma inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-gamma suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning -6368 to +125 bp in the absence and presence of the intronic enhancer and stimulated activity of the gamma-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1 alpha-binding activity in nuclear extracts. These responses to IFN-gamma were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1 alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma 2A)- and Stat1 alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1 alpha, but not by either Stat1 alpha Y701F or Stat1 beta. Functional analysis using deletion constructs showed that the IFN-gamma response was retained in the COL2A1 core promoter region spanning -45 to +11 bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-gamma persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN-gamma requires Jak1, Jak2 and Stat1 alpha and suggest that this response involves indirect interaction of activated Stat1 alpha with the general transcriptional machinery that drives constitutive COL2A1 expression.

T cell-specific expression of the murine CD3delta promoter.

T cell-specific expression of human and mouse CD3delta is known to be governed by an enhancer element immediately downstream from the gene. Here we demonstrate by transgenic and in vitro studies that the murine CD3delta (mCD3delta) promoter prefers to be expressed in cells of the T lineage. Deletion analyses of a promoter segment (-401/+48 bp) followed by transient transfections indicate that upstream elements between -149 and -112 bp contribute to full expression of the gene. Furthermore, a core promoter region -37/+29 appears to contribute to a T cell specificity. Using substitution mutant scanning, four positive and one negative regulatory elements were found within the mCD3delta core promoter. The first two positive elements comprise two classical initiator-like sites, which recruit TFII-I, whereas a third contains a functional Ets binding site. Immediately adjacent to the observed transcription start site is a negative element that utilizes the transcription regulator YY1. The last positive regulatory element contains a potentially functional CREB binding site and the minor transcriptional start site. Because NERF-2, Elf-1, and Ets-1 are expressed preferentially in lymphocytes and because, in addition, YY1 represses the promoter activity strongly in non-T cells, we conclude that the combination of these transcription factors contributes to the T cell-specific expression pattern of mouse CD3delta.

Differentiation regulates interleukin-1beta-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase.

Chondrocyte dedifferentiation has been noted in osteoarthritic cartilage, but the contribution of this phenomenon is poorly understood. Interleukin (IL)-1beta, the major pro-inflammatory cytokine found in osteoarthritic synovial fluid, induces the dedifferentiation of cultured articular chondrocytes, whereas E-series prostaglandins (PGE) are capable of inducing cell differentiation. Since PGE(2) synthesis is up-regulated by IL-1beta, we addressed the question of whether the state of chondrocyte differentiation may influence the production of IL-1-induced PGE(2) by modulating cyclooxygenase (COX)-2 expression. Immortalized human articular chondrocytes, (tsT/AC62) cultured in monolayer after passage through alginate matrix (alg+) produced 5-fold greater amounts of PGE(2) than continuous monolayer cultures (alg-) after stimulation with IL-1beta. Moreover, IL-1beta induced COX-2 expression at 0.01 ng/ml in (alg+) cells, whereas a 100-fold higher dose of cytokine was necessary for stimulation in (alg-) cells. SB203580, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor, completely abolished the IL-1beta-induced COX-2 mRNA. Overexpression of p38 MAPK induces a COX-2 reporter, whereas overexpression of dominant negative p38 MAPK represses IL-1beta-induced promoter expression. Interestingly, IL-1beta-induced p38 MAPK activity was greatly enhanced in (alg+) compared with (alg-) cells. Our results suggest that differentiated articular chondrocytes are highly responsive to IL-1beta and that p38 MAPK mediates this response by inducing COX-2 gene expression.