The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1-70K and SR proteins and therefore may acquire new functions. INTRODUCTIONSplicing factors are found within the nucleus both in a diffuse form and in distinct domains termed interchromatin granules (IG) or "speckles" (Spector, 1993). These functional compartments are spatially dynamic with regard to movement and composition (Eils et al., 2000). Splicing factors within IGs are highly mobile and are constantly associating and dissociating from their compartments (Phair and Misteli, 2000). The mAb, B92, which recognizes the polypyrimidine tract binding protein (PTB)-associated splicing factor (PSF), produces typical specked nuclear pattern in immunostaining. These PSF speckles disappear during granulocytic differentiation (Lee et al., 1996;Shav-Tal et al., 2000). We recently showed that this disappearance is associated with partial degradation (Shav-Tal et al., 2000) and by the formation of alternative nuclear structures (Shav-Tal et al., 2001). The present study indicates that PSF speckles disappear also through a different mechanism, involving conformational changes that cause breakdown of PSF speckles and relocalization of the molecule in the nucleus. The functional nature of speckles is a matter of some controversy (for review, see Park and De Boni, 1999). These structures have been suggested to play a role in storage and recycling of splicing factors (Jimenez-Garcia and Spector, 1993), whereas a portion may act as reservoirs for the recruitment to active sites of transcription (Huang and Spector, 1996;Zeng et al., 1997). This view does not predict a direct involvement of speckles in pre-mRNA splicing. On the other hand, it has been shown that nascent mRNA transcripts and transcription sites are closely associated with speckles, that transcription occurs at the surface of speckles (Clemson and Lawrence, 1996), and that microinjected pre-mRNAs have high affinity to speckles (Wang et al., 1991). It has thus been proposed that speckles can act coordinately in transcription and splicing and that pre-mRNA splicing can take place bo...

Activin A, a member of the transforming growth factor ␤ (TGF␤) superfamily, blocks interleukin (IL)-6 biological functions. The molecular basis of the influence of this TGF␤ signaling on the IL-6 receptor triggered cascade is unknown. We studied IL-6-induced secretion of the acute phase protein haptoglobin by hepatoma cells. Overexpression of the C/EBP␤ gene, a downstream effector in the IL-6 pathway, activated transcription from the haptoglobin promoter. This was abolished by either a constitutively active form of activin A type IB receptor (CAactRIB) or by a combination of Smad3 and Smad4. Similarly, Smads abolished transcriptional activation by co-stimulation with IL-6 and STAT3. The transcription co-activator p300 partially overcame the suppressive effect of Smads. Electrophoretic mobility shift assays indicated that C/EBP␤ binding to haptoglobin promoter DNA was reduced by over-expression of CAactRIB and Smad4. We thus show that Smad proteins operate as transcription inhibitors on target genes of the IL-6 induced pathway. The effect of Smads is exerted on components of the transcription activation complex and may also involve interference with DNA binding. This study thus depicts molecular sites of interaction between the TGF␤ superfamily and the IL-6 signaling cascades.The execution of activin A biological functions depends upon association of the cytokine with type I and type II serine/ threonine kinase membrane receptors (1, 2). Activin A first binds to activin receptor (ActR) 1 type II. This is followed by recruitment into the complex and phosphorylation of activin receptor type IB (actRIB), which is responsible for downstream signal transduction. Smad proteins are effectors of intracellular signal transduction of members of the TGF␤ superfamily (2-5). The mediators of activin signaling are receptor-activated Smad2 and Smad3 (6), which are present in the cytoplasm in a monomeric form. Following activation, they undergo homodimerization and then heterodimerize with Smad4 (CoSmad) that lacks the C-terminal phosphorylation motif and is therefore not activated by type I receptors (6, 7). The complex of Smad4 and receptor-activated Smads translocates to the nucleus and binds to Smad binding elements in specific promoters (8 -10). Smad3 and Smad4 bind through their conserved MH1 domain to the palindromic sequence GTCTAGAC (11, 12) and to CAGA-like sequences in the human plasminogen activator inhibitor-1 (PAI-1) and junB genes (13-15). Smads cooperate with various transcription factors such as c-Fos and c-Jun in binding to AP-1 sites (16). Smads also complex with Forkhead activin signal transducer (FAST) proteins for efficient binding to specific promoters (17) and associate with the closely related transcriptional co-activators p300 and CBP. The latter interact with a variety of transcription factors and thereby link these factors to the basal transcription machinery (18 -20). A hallmark in Smad signaling is therefore their interaction with other transcription regulators. Engagement of the activin A receptors...

Objective. Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is manifested by neurologic deficits and psychiatric disorders. The aim of this study was to examine SLE-associated CNS pathology in lupus-prone (NZB ؋ NZW)F 1 (NZB/ NZW) mice, and to evaluate the ameliorating effects of treatment with a tolerogenic peptide, hCDR1 (human first complementarity-determining region), on these manifestations.Methods. Histopathologic analyses of brains from lupus-prone NZB/NZW mice treated with vehicle, hCDR1, or a control scrambled peptide were performed. The messenger RNA expression of SLE-associated cytokines and apoptosis-related molecules from the hippocampi was determined. Anxiety-like behavior was assessed by open-field tests and dark/light transfer tests, and memory deficit was assessed using a novel object recognition test.Results. Infiltration was evident in the hippocampi of the lupus-afflicted mice, and the presence of CD3؉ T cells as well as IgG and complement C3 complex deposition was observed. Furthermore, elevated levels of gliosis and loss of neuronal nuclei immunoreactivity were also observed in the hippocampi of the mice with lupus. Treatment with hCDR1 ameliorated the histopathologic changes. Treatment with hCDR1 down-regulated the high expression of interleukin-1␤ (IL-1␤), IL-6, IL-10, interferon-␥, transforming growth factor ␤, and the proapoptotic molecule caspase 8 in the hippocampi of the mice with lupus, and up-regulated expression of the antiapoptotic bcl-x L gene. Diseased mice exhibited increased anxiety-like behavior and memory deficit. Treatment with hCDR1 improved these parameters, as assessed by behavior tests.Conclusion. Treatment with hCDR1 ameliorated CNS pathology and improved the tested cognitive and mood-related behavior of the mice with lupus. Thus, hCDR1 is a novel candidate for the treatment of CNS lupus.

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SummarySystemic lupus erythematosus (SLE) is an autoimmune disease that involves dysregulation of B and T cells. A tolerogenic peptide, designated hCDR1, ameliorates disease manifestations in SLE-afflicted mice. In the present study, the effect of treatment with hCDR1 on the CD74/macrophage migration inhibitory factor (MIF) pathway was studied. We report here that B lymphocytes from SLE-afflicted mice express relatively elevated levels of CD74, compared with B cells from healthy mice. CD74 is a receptor found in complex with CD44, and it binds the pro-inflammatory cytokine MIF. The latter components were also up-regulated in B cells from the diseased mice, and treatment with hCDR1 resulted in their down-regulation and in reduced B-cell survival. Furthermore, up-regulation of CD74 and CD44 expression was detected in brain hippocampi and kidneys, two target organs in SLE. Treatment with hCDR1 diminished the expression of those molecules to the levels determined for young healthy mice. These results suggest that the CD74/MIF pathway plays an important role in lupus pathology.