PTHrP,PTHr, and FGFR3 are involved in the process of endochondral ossification in human osteophytes

To elucidate the process of endochondral ossification in human osteophytes we have studied the expression of parathyroid hormone-related protein (PTHrP), its receptor (PTHr), and fibroblast growth factor receptor 3 (FGFR3). Osteophytes from patients undergoing total knee replacement ( n=13), and fetal growth plate cartilages ( n=4) were processed for safranin O staining and immunohistochemistry. Chondrocytes and their matrix were preferentially stained for PTHrP in the middle and deep zones of the osteophytes examined. Ossified areas did not show a positive staining. In fetal joints the cartilaginous surface and the perichondrium as well as the osteoblasts in the trabecular bone were positive. PTHr was expressed at large in chondrocytes and osteoblasts of all osteophytes and fetal joints. Cells of the perichondrium were also positive. The FGFR3 antibody stained only single chondrocytes in some osteophytes, and groups of cells in others. In fetal samples, chondrocytes of the proliferating and the hypertrophic zone showed staining for FGFR3. This is the first report on the expression of PTHrP, PTHr, and FGFR3 in human osteophytes. As in fetal joints these mediators might regulate proliferation and differentiation of chondrocytes playing an important role in osteo(chondro)phyte growth.     

Molecular mechanisms of L-selectin-induced co-localization in rafts and shedding [corrected

Leukocyte recruitment to lymph nodes or inflammatory sites is regulated by adhesion and activation. L-selectin (CD62L) is expressed on leukocytes and mediates tethering and rolling of leukocytes on endothelial cells. Upon stimulation L-selectin is down-regulated by proteolytic cleavage but the molecular mechanisms regulating this shedding step are poorly defined. To study intracellular mechanisms, we induced shedding of L-selectin by cross-linking with an immobilized L-selectin antibody (Dreg56) in Jurkat cells. The loss of surface expression was quantitated by flow cytometry and the increase of soluble L-selectin was determined by Western blot analysis. We find that Jurkat and p56(lck)-deficient JCaM1.6 cells released L-selectin to similar extent (18+/-4% and 17+/-3%, respectively) and revealed comparable inhibition with the src-tyrosine kinase inhibitor PP2. Glutathione (GSH), an inhibitor of the neutral sphingomyelinase, PD98059, a MAP-kinase (MAP-K) inhibitor and metalloprotease inhibitors (MPI) (TAPI, Ro 31-9790, and BB-3103) reduced significantly L-selectin-induced shedding by 60-80%. In Jurkat cells, L-selectin was present in Triton X-100 insoluble membrane rafts and was constitutively tyr-phosphorylated. Dreg56 cross-linking enhanced phosphorylation and recruitment of L-selectin into rafts which was significantly decreased by pretreatment of cells with PD98059. We conclude, that the metalloproteinase-mediated cleavage of L-selectin from cell surface is triggered by intracellular signaling pathways that are independent of p56(lck) tyrosine kinase activity, but require other tyrosine kinases and the neutral sphingomyelinase. The cleavage of L-selectin might involve membrane rafts as signaling platform.     

Curvature in hand movements as a result of visual misjudgements of direction

The path that our hand takes when moving from one position to another is often slightly curved. Part of this curvature is caused by perceptual errors. We examine here whether this is so for the influence that a surface's orientation has on the approaching hand's path. When moving our hand towards a point on a surface we tend to follow a path that makes the final approach more orthogonal to the surface at that point. Doing so makes us less sensitive to imperfections in controlling our movements. Here we show that this tendency is also present when moving towards a point along an edge of a drawing of an oriented bar. The influence of the bar's orientation is no smaller when people are explicitly asked to move as straight as possible, than when they are instructed to move as fast as possible. The bar's orientation also influences perceptual judgements of a straight path, but this influence is only as large as it is on the curvature of the hand's path for judgements of the direction from the hand's initial position to the target. We conclude that the influence of the bar's orientation on the curvature of the hand's path is caused by a misperception of the initial direction in which the hand has to move to reach the target.     

Chromosomes are predominantly located randomly with respect to each other in interphase human cells

To test quantitatively whether there are systematic chromosome-chromosome associations within human interphase nuclei, interchanges between all possible heterologous pairs of chromosomes were measured with 24-color whole-chromosome painting (multiplex FISH), after damage to interphase lymphocytes by sparsely ionizing radiation in vitro. An excess of interchanges for a specific chromosome pair would indicate spatial proximity between the chromosomes comprising that pair. The experimental design was such that quite small deviations from randomness (extra pairwise interchanges within a group of chromosomes) would be detectable. The only statistically significant chromosome cluster was a group of five chromosomes previously observed to be preferentially located near the center of the nucleus. However, quantitatively, the overall deviation from randomness within the whole genome was small. Thus, whereas some chromosome-chromosome associations are clearly present, at the whole-chromosomal level, the predominant overall pattern appears to be spatially random.     

Differential requirement for NF-kappaB-inducing kinase in the induction of NF-kappaB by IL-1beta,TNF-alpha,and Fas

In this study, weexamined the role of the nuclear factor-B (NF-B)-inducing kinase(NIK) in distinct signaling pathways leading to NF-B activation. Weshow that a dominant-negative form of NIK (dnNIK) delivered byadenoviral (Ad5dnNIK) vector inhibits Fas-induced IBphosphorylation and NF-B-dependent gene expression in HT-29 and HeLacells. Interleukin (IL)-1- and tumor necrosis factor-(TNF-)-induced NF-B activation and B-dependent gene expressionare inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells.Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF--inducedapoptosis at an early time point. However, cytokine- andFas-induced signals to NF-B are finally integrated by the IBkinase (IKK) complex, since IB phosphorylation, NF-B DNAbinding activity, and IL-8 gene expression were strongly inhibited inHT-29 and HeLa cells overexpressing dominant-negative IKK(Ad5dnIKK). Our findings support the concept that cytokine signalingto NF-B is redundant at the level of NIK. In addition, this studydemonstrates for the first time the critical role of NIK and IKK inFas-induced NF-B signaling cascade.

     

Heat shock protein expression and anti-heat shock protein reactivity in renal cell carcinoma

Heat shock proteins (HSP) are families of highly conserved proteins which are induced in cells and tissues upon exposure to extreme conditions causing acute or chronic stress. They exhibit distinct functions and have been implicated in the pathogenesis of a number of diseases, including cancer. A causal relationship between HSP expression and immunogenicity has been demonstrated in murine and human tumors and is also associated with the immune response. In order to investigate the correlation of HSP expression and their immunogenic potential in renal cell carcinoma (RCC), we here analyzed (i) the protein expression profile of various members of the HSP family in untreated and interferon (IFN)-gamma treated RCC cell lines as well as normal kidney epithelium, and (ii) the anti-heat shock protein reactivity in sera derived from RCC patients and healthy controls using proteomics-based techniques. A heterogeneous expression pattern of members of the HSP families was demonstrated in RCC cell lines and in cells representing normal renal epithelium. In some cases the expression rate is moderately altered by IFN-gamma treatment. In addition, a distinct anti-heat shock protein reactivity could be detected in autologous and allogeneic sera from RCC patients and healthy controls. These data suggest that HSP play a role in the immunogenicity of RCC and thus might be used for the design of immunization strategies to induce a potent antitumor response in this disease.     

Dominant-negative TAK1 induces c-Myc and G(0) exit in liver

Transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1), a serine/threonine kinase, is reported to function in the signaling pathways of TGF-beta, interleukin 1, and ceramide. However, the physiological role of TAK1 in vivo is largely unknown. To assess the function of TAK1 in vivo, dominant-negative TAK1 (dnTAK1) was expressed in the rat liver by adenoviral gene transfer. dnTAK1 expression abrogated c-Jun NH(2)-terminal kinase and c-Jun but not nuclear factor (NF)-kappaB or SMAD activation after partial hepatectomy (PH). Expression of dnTAK1 or TAM-67, a dominant-negative c-Jun, induced G(0) exit in quiescent liver and accelerated cell cycle progression after PH. Finally, dnTAK1 and TAM-67 induced c-myc expression in the liver before and after PH, suggesting that G(0) exit induced by dnTAK1 and TAM-67 is mediated by c-myc induction.     

Autocrine expression of activated transforming growth factor-beta(1) induces apoptosis in normal rat liver

The aim of this study was to determine the differential effects of latent and activated transforming growth factor (TGF)-beta(1) in growth control of normal and proliferating hepatocytes in vivo. Rats were injected with adenoviruses expressing control transgenes (Ctrl), latent TGF-beta(1) [TGF-beta(L)], or activated TGF-beta(1) [TGF-beta(A)]. Additional animals underwent two-thirds partial hepatectomy (PH) 24 h after injection. Increased hepatocyte apoptosis was observed in TGF-beta(A)-injected but not TGF-beta(L)-injected animals 24 h postinjection (10.5%) compared with Ctrl animals (0.37%). The percent of apoptotic cells increased to 32.1% in TGF-beta(A)-injected animals 48 h after injection. Furthermore, TGF-beta(A)-injected rats did not survive 24 h after PH. Four hours after PH, 0.25 and 14.1% apoptotic hepatocytes were seen in Ctrl- and TGF-beta(A)-injected rats, respectively. TGF-beta(A)-induced apoptosis in primary rat hepatocytes was blocked with a pancaspase inhibitor. Thus autocrine expression of TGF-beta(A) but not TGF-beta(L) induces hepatocyte apoptosis in the normal rat liver. Rats overexpressing TGF-beta(A) do not survive two-thirds PH due to hepatic apoptosis. Thus activation of TGF-beta(1) may be a critical step in the growth control of normal and proliferating rat hepatocytes.