The transcription factor AP-2ɛ regulates CXCL1 during cartilage development and in osteoarthritis

SummaryObjectiveRecently, the transcription factor AP-2ɛ was shown to be a regulator of hypertrophy in cartilage and to be differentially expressed in osteoarthritis (OA). However, the only known target gene of AP-2ɛ up to date is integrin alpha10. To better characterize the function of AP-2ɛ in cartilage we screened for additional target genes.DesignPromoter analysis, ChIP-assays and electrophoretic mobility shift assay were used to characterize the regulation of a new AP-2ɛ target gene in detail.ResultsIn this study, we determined the chemokine CXCL1, already known to be important in osteoarthritis (OA), as a new target gene of AP-2ɛ. We could confirm that CXCL1 is expressed in chondrocytes and significantly over-expressed in OA-chondrocytes. Transient transfection of chondrocytes with an AP-2ɛ expression construct led to a significant increase of the CXCL1 mRNA level in these cells. We identified three potential AP-2 binding sites within the CXCL1 promoter and performed luciferase assays, indicating that an AP-2 binding motif (AP-2.2) ranging from position −135 to −144bp relative to the translation start is responsive to AP-2ɛ. This result was further addressed by site-directed mutagenesis demonstrating that activation of the CXCL1 promoter by AP-2ɛ is exclusively dependent on AP-2.2. Chromatin immunoprecipitation and electromobility shift assays confirmed the direct binding of AP-2ɛ to the CXCL1 promoter in OA-chondrocytes at this site.ConclusionThese findings revealed CXCL1 as a novel target gene of AP-2ɛ in chondrocytes and support the important role of AP-2ɛ in cartilage

Expression, function and clinical relevance of MIA (Melanoma Inhibitory Activity)

Despite its ambiguous name the protein melanoma-inhibitory-activity (MIA) was identified as a key molecule involved in progression and metastasis of malignant melanomas. Therefore, in this review we intend to update the current knowledge on expression patterns, transcriptional regulation, function and clinical relevance of MIA. Furthermore, we will cover the recently discovered MIA homologous proteins OTOR/MIAL, MIA 2 and TANGO. In order to identify autocrine growth-regulatory factors secreted by melanoma cells, MIA was purified and cloned. Subsequent analyses of non-neoplastic tissues revealed specific MIA expression patterns in cartilage. In neoplastic tissues MIA expression was detected in malignant melanomas, in chondrosarcomas and less frequently in a variety of diff e r e n t adenocarcinomas including breast and colon cancers. For melanoma cells and chondrocytes it was shown that regulation of expression pattern was controlled on the level of mRNA transcription by defined transcription factors. Evidence obtained from in vitro and in vivo experiments indicated that MIA plays an important functional role in melanoma metastasis and invasion. A number of studies from different laboratories evaluated MIA as a highly specific and sensitive marker, clinically useful for follow-up and therapy-monitoring of patients with malignant melanomas. In addition, preliminary data suggests a further potential application as a surrogate marker for measuring cartilage damage in rheumatoid arthritis. Recently, it has become evident that MIA belongs to a gene family of four homologous proteins, MIA, OTOR (FDP, MIAL), MIA 2 and TANGO. Determination of the three-dimensional structure in solution identified MIA as the first member of this novel family of secreted, extracellular proteins adopting an SH3 domain-like fold. The data suggest specific protein-protein interactions with components of the extracellular matrix and possibl

Transcription factors involved in development and progression of malignant melanoma

Up to date many genes are known to be deregulated in tumor development and progression. Genes important in tumorigenesis belong to families such as proteases, kinases and receptors. However, an important family of proteins is rarely discussed: the mediators of transcriptional control, the transcription factors. Usually, changes in transcription factor expression or activity can lead to more than just one downstream modification, as transcription factors are higher, thinking in a hierarchical way of expression control. In this review we summarize the role of the transcription factors AP-1, AP-2alpha, CREB, CtBP, ETS-1, HMGB1, LEF/TCF/ß-catenin, MITF, NF?B, PAX3, SKI, Snail and STAT in carcinogenesis focusing on melanoma development and progressio

Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions

Analyses of protein interactions are fundamental for the investigation of molecular mechanisms responsible for cellular processes and diseases, as well as for drug discovery in the pharmaceutical industry. The present study details the development of a fluorescence polarization assay using melanoma inhibitory activity (MIA) protein–binding compounds and studies of the binding properties of this protein. Since they are dependent on the the lifetime of the fluorescent label, currently available fluorescence polarization assays can only determine interactions with either high– or low–molecular weight interaction partners. Our new approach eliminates this limitation by immobilizing a known binding partner of MIA protein to a well plate and by labeling the target protein using luminescent transition metal labels such as Ru(bpy)3 for binding studies with both high– and low–molecular weight interaction partners. Due to the use of a functionalized surface, we termed our concept heterogeneous transition metal–based fluorescence polarization (HTFP) assay. The assay’s independence from the molecular weight of potential binding partners should make the technique amenable to investigations on subjects as diverse as multimerization, interactions with pharmacophores, or binding affinity determination

Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks

In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm3). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools