The uninduced Drosophila hsp70 gene is poised for rapid activation. Here we examine the rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms.Recent studies of posttranslational modifications of, and factor interactions with, the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) have implicated this region of Pol II in productive transcriptional elongation and the coupling of transcription and pre-mRNA processing (for reviews see references 32 and 37). The CTD is highly conserved among eukaryotes, containing multiple repeats of a consensus heptad YSPTSPS; mammalian Pol II CTD consists of 52 repeats, yeast consists of 25 to 26 repeats, while Drosophila has 42 repeats (1, 6). This large flexible arm of Pol II appears to serve as a docking site for, or to stimulate the recruitment of, an orchestrated assembly of factors involved in pre-mRNA capping, splicing, and 3Ј polyadenylation at different stages in production of the nascent transcript (5,9,13,14,20,26). One way this coordination has been proposed to occur is through the known phosphorylation of serines 2 and 5 (Ser2-P and Ser5-P, respectively) of the heptad repeat (52, 56). Cdk7, the kinase subunit of general transcription factor TFIIH, has been shown to phosphorylate the CTD at Ser5, an event proposed to occur early in the transcription cycle (12,24,57). This in turn appears to influence the association and activity of the capping machinery (5,15,18,27,28,42,45). Positive transcription elongation factor b (P-TEFb) is able to phosphorylate the CTD at Ser2 and, under certain conditions, Ser5 (25, 38, 57). P-TEFb is a kinase composed of the proteins cyclin T (CycT) and cdk9 and is known to be recruited upon gene activation, overcoming the negative effects of factors like Spt5 and negative elongation factor (50) and aiding in the transition from transcription initiation to elongation (36). Cdk8, a component of the coactivator complex Mediator, h...

Objective. Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1␤ (IL-1␤) signaling in chondrocytes.Methods. Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. The Hsp90 inhibitor geldanamycin (50 nM, 100 nM, or 500 nM) was used to assess age-related responses to Hsp90 with concurrent IGF-1 or IL-1␤ stimulation of chondrocytes. Quantitative real-time PCR was used to measure COL2A1 and matrix metalloproteinase 13 (MMP13) gene expression; Western blot analysis was performed to determine the phosphorylation status of p42/44 and Akt/protein kinase B.Results. The effects of Hsp90 inhibition with geldanamycin were concentration dependent. Inhibition of Hsp90 with 100 nM or 500 nM geldanamycin blocked IGF-1-induced cell proliferation, Akt and p42/44 activation, and COL2A1 expression. Basal and IL-1␤-induced up-regulation of MMP13 mRNA was blocked by all concentrations of geldanamycin tested. Gain-offunction assays with Hsp90 resulted in increased expression of MMP13 mRNA.Conclusion. These results suggest that Hsp90 is involved in opposing signaling pathways of cartilage homeostasis, and that catabolic responses are more sensitive to Hsp90 inhibition than are anabolic responses. Further studies are needed to determine the role of Hsp90 inhibition in osteoarthritis in order to assess its potential as a therapeutic target.

Small GTPases regulate the cytoskeleton and numerous other cellular functions. In this study, the role of Rho GTPase was examined in articular chondrocytes. Chondrocytes grown in monolayer were treated with interleukin-1a (IL-1a), insulin-like growth factor-I (IGF-I), C3 Transferase, Y27632, or transfected with Rho wild type or two constitutively active mutants of Rho (Q63L and G14V). Quantitative PCR was used to determine changes in matrix metalloproteinase-13 (MMP-13), collagen types IIB (COL2A1) and type I (COL1A1), aggrecan (AGG), and SOX-9 gene expression. Affinity assays were performed to measure endogenous GTP-bound Rho, and confocal microscopy was used to assess changes in organization of the actin cytoskeleton. IL-1a and RhoG14V increased cytoplasmic actin stress fiber formation, which was blocked by C3 Transferase, and Y27632. IL-1a treatment also increased Rho activity. Conversely, IGF-I lead to formation of a cortical rim of actin and decreased Rho activity. Inhibition of Rho signaling with C3 Transferase significantly decreased Rho activity and returned IL-1a-induced Rho activity to a level not different from control. C3 Transferase treatment also increased mRNA expression of AGG, COL2A1, and SOX-9, and decreased expression of MMP-13. Expression of RhoQ63L or RhoG14V resulted in increased MMP-13 expression; however, inhibition of Rho with Y27632 was unable to inhibit IL-1a-induced MMP-13 expression. Together, these results indicate a role for increased Rho activity in mediation of chondrocyte catabolic signaling pathways. [3][4][5][6][7][8][9] Changes in cell morphology and organization of the cytoskeleton have also been associated with OA. 10,11 Defining basic intracellular signaling pathways that regulate chondrocyte phenotypic expression and morphology might further elucidate the pathogenesis of OA and reveal novel therapeutic targets.One signaling pathway that has been implicated in regulation of articular chondrocyte morphology and phenotype is the Rho subfamily of small GTPases. [4][5][6][7][8][9] The Rho subfamily of small GTPases are 21-22-kDa proteins involved in many cellular processes such as regulation of actin cytoskeleton and morphology, cell cycle progression, and integrin assembly (see Schwartz 12 for review). The Rho subfamily includes Cdc42, Rac, and Rho, which cycle between active GTP-bound and inactive GDP-bound states. Activation of Rho GTPases results in reorganization of the cytoskeleton, and only when they are in their active form can they affect downstream targets. 13 The three variants of Rho, RhoA, RhoB, and RhoC, share over 85% homology and have nearly indistinguishable actions and interactions, so the more general term Rho will be used herein. 14 The roles of small GTPases in articular chondrocytes have been partially elucidated. 8,9,15 Treatment with insulin-like growth factor-I (IGF-I) reduces actin stress fibers, diminishes activation of Cdc42 and Rac, and is associated with preservation of the normal chondrocyte phenotype. 8 In chondrocytes, assembly of an...

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