Analysis of heat shock proteins and cytokines expressed during early stages of osteoarthritis in a mouse model

SummaryObjective:Osteoarthritis (OA) is a debilitating disease of the joints. The joints of affected individuals are characterized by a progressive degeneration of articular cartilage leading to inflammation and pain. The expression of heat shock proteins (HSPs) is a ubiquitous self-protective mechanism of all cells under stress, furthermore, the synovium of osteoarthritic individuals contains high levels of cytokines. This study seeks to establish the role of HSPs and cytokines in OA.Methods:We have investigated the presence of HSPs and cytokines in articular cartilage during early stages of OA in a mouse that is known to develop spontaneous OA lesions (C57 black mouse). The articular cartilage from closely related mice (C57BL/6) was used as control. Messenger RNAs (mRNAs) for HSPs (HSP32, HSP47, HSP60, HSP70, HSP84 and HSP86) and cytokines [interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)] were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results:The mRNA levels of HSP47, HSP70, HSP86, IL-6, and IFN-γ were up-regulated in the cartilage of C57 black mice, whereas, the level of expression of HSP32, HSP60, HSP84 and IL-1β remained unchanged. Furthermore, the expression of IL-1β, IL-6, TNF-α and IFN-γ mRNA was associated with expression of HSP60, HSP47, HSP70 and HSP70/HSP86 mRNA, respectively.Conclusions:The findings in this study suggest that chondrocytes are conditioned under non-physiological stress during early stages of OA, In addition, among HSPs, HSP70 was associated with two different highly expressed cytokines in C57 black mice, indicating the possible role of HSP70 as a characteristic indicator of early stage of OA

Characterization of the role of the proximal sequence element (PSE) and the TATA box in the human U6 small nuclear RNA (snRNA) gene transcription

Vita.Transcription of vertebrate U6 snRNA genes by RNA polymerase III requires two sequence elements in the proximal promoter region: the PSE (proximal sequence element, found in snRNA promoters transcribed by RNA polymerase II) and the TATA element (found in many mRNA promoters). The locations of the PSE and the TATA box are important determinants for transcriptional start site selection in their respective RNA polymerase II promoters. In vertebrate U6 genes the PSE and the TATA elements are located in approximately the same positions as in the polymerase II transcribed genes. However, their respective roles in initiation site selection, transcriptional efficiency and polymerase specificity are not known. I have analyzed the effects of spacing changes between the PSE and the TATA element, and between the two elements and the normal U6 start site on human U6 gene transcription in vitro and in transfected cells. My results discount the possibility that the location of either the PSE or the TATA element, by itself, dictates efficient selection of a transcriptional start site. Instead, I suggest that the two elements form a compound promoter element whose location dictates the start site of transcription from the hum an U6 gene promoter. Spacing changes between the PSE and the TATA element result in greatly reduced levels of transcription. These observations suggest the possibility that the factors that bind these two elements interact with each other. To test this hypothesis, I further investigated the possible interaction of the PSE and TATA binding proteins (PBP and TBP) when bound to normal or mutant U6 proximal promoters using a gel mobility shift assay. I detect a complex containing both PBP and TBP bound to the wild-type U6 promoter. Efficient formation of the triple complex is dependent on the presence of the PSE and the TATA box on the template DNA. Furthermore, U6 mutant promoters containing an increased spacing between the PSE and TATA box of 5 or 10 bp are impaired in the ability to form a complex that includes TBP. I infer from these results that PBP and TBP interact when their binding sites are properly positioned in a U6 gene promoter

Characterization of the role of the proximal sequence element (PSE) and the TATA box in the human U6 small nuclear RNA (snRNA) gene transcription

Vita.Transcription of vertebrate U6 snRNA genes by RNA polymerase III requires two sequence elements in the proximal promoter region: the PSE (proximal sequence element, found in snRNA promoters transcribed by RNA polymerase II) and the TATA element (found in many mRNA promoters). The locations of the PSE and the TATA box are important determinants for transcriptional start site selection in their respective RNA polymerase II promoters. In vertebrate U6 genes the PSE and the TATA elements are located in approximately the same positions as in the polymerase II transcribed genes. However, their respective roles in initiation site selection, transcriptional efficiency and polymerase specificity are not known. I have analyzed the effects of spacing changes between the PSE and the TATA element, and between the two elements and the normal U6 start site on human U6 gene transcription in vitro and in transfected cells. My results discount the possibility that the location of either the PSE or the TATA element, by itself, dictates efficient selection of a transcriptional start site. Instead, I suggest that the two elements form a compound promoter element whose location dictates the start site of transcription from the hum an U6 gene promoter. Spacing changes between the PSE and the TATA element result in greatly reduced levels of transcription. These observations suggest the possibility that the factors that bind these two elements interact with each other. To test this hypothesis, I further investigated the possible interaction of the PSE and TATA binding proteins (PBP and TBP) when bound to normal or mutant U6 proximal promoters using a gel mobility shift assay. I detect a complex containing both PBP and TBP bound to the wild-type U6 promoter. Efficient formation of the triple complex is dependent on the presence of the PSE and the TATA box on the template DNA. Furthermore, U6 mutant promoters containing an increased spacing between the PSE and TATA box of 5 or 10 bp are impaired in the ability to form a complex that includes TBP. I infer from these results that PBP and TBP interact when their binding sites are properly positioned in a U6 gene promoter

Adhesion of perichondrial cells to a polylactic acid scaffold

The number of chondrogenic cells available locally is an, important factor in the repair process for cartilage defects. Previous studies demonstrated that the number of transplanted rabbit perichondrial cells (PC) remaining in a cartilage defect in vivo, after being carried into the site in a polylactic acid (PLA) scaffold, declined markedly within two days. This study examined the ability of in vitro culture of PC/PLA constructs to enhance subsequent biomechanical stability of the cells and the matrix content in an in vitro screening assay. PC/PLA constructs were analyzed after 1 h, 1 and 2 weeks of culture. The biomechanical adherence of PC to the PLA scaffold was tested by subjecting the PC/PLA constructs to a range of flow velocities (0.25-25 mm/s), spanning the range estimated to occur under conditions of construct insertion in vivo. The adhesion of PC to the PLA carrier was increased significantly by 1 and 2 weeks of incubation, with 25 mm/s flow causing a 57% detachment of cells after 1 h of seeding, but only 7% and 16% after I and 2 weeks of culture, respectively (p < 0.001). This adherence was associated with marked deposition of glycosaminoglycan and collagen. These findings suggest that pre-incubation of PC-laden PLA scaffolds markedly enhances the stability of the indwelling cells. (C) 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved