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Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

Using a novel model of rheumatoid arthritis, we identified a critical role for reduced tissue-resident macrophages., Little is known about the mechanisms regulating the transition of circulating monocytes into pro- or anti-inflammatory macrophages in chronic inflammation. Here, we took advantage of our novel mouse model of rheumatoid arthritis, in which Flip is deleted under the control of a CD11c promoter (HUPO mice). During synovial tissue homeostasis, both monocyte-derived F4/80int and self-renewing F4/80hi tissue–resident, macrophage populations were identified. However, in HUPO mice, decreased synovial tissue–resident macrophages preceded chronic arthritis, opened a niche permitting the influx of activated monocytes, with impaired ability to differentiate into F4/80hi tissue–resident macrophages. In contrast, Flip-replete monocytes entered the vacated niche and differentiated into tissue-resident macrophages, which suppressed arthritis. Genes important in macrophage tissue residency were reduced in HUPO F4/80hi macrophages and in leukocyte-rich rheumatoid arthritis synovial tissue monocytes. Our observations demonstrate that the macrophage tissue–resident niche is necessary for suppression of chronic inflammation and may contribute to the pathogenesis of rheumatoid arthritis.

Objective: Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP (FLICE-like inhibitory protein) is necessary for the differentiation and/or survival of macrophages. We also identified that FLIP is highly expressed in RA synovial macrophages. This study was performed to determine if the reduction of Flip in macrophages would reduce synovial tissue macrophages and ameliorate serum transfer induced arthritis (STIA). Methods: Mice with Flip deleted in myeloid cells (Flipf/fLysMc/+ mice) and littermate controls were employed. STIA was induced by intraperitoneal injection of K/BxN serum. Arthritis was evaluated by clinical score and change in thickness of the ankles. Joints were examined by histology and immunohistochemistry. Cells were isolated from the ankles and bone marrow and examined by flow cytometry, qRT-PCR or Western blot. Results: In contrast to expectations, Flipf/fLysMc/+ mice developed more severe arthritis early in the clinical course, however the peak arthritis was attenuated and the resolution phase more complete. Prior to the induction of STIA, tissue resident macrophages were reduced. Further at day 9 post arthritis induction the number of F4/80hi macrophages in the joints of the Flipf/fLysMc/+ mice was not decreased, but increased. Flip was reduced in the F4/80hi macrophages in the ankles of the Flipf/fLysMc/+ mice, while F4/80hi population expressed an anti-inflammatory phenotype in both the Flipf/fLysMc/+ and control mice. Conclusions: These observations suggest that reduction of FLIP in macrophages, by increasing the number of anti-inflammatory macrophages, may be an effective therapeutic approach to suppress inflammation, depending upon the disease stage. This article is protected by copyright. All rights reserved.

The essential role of mechanical signals in regulating the function of living cells is universally observed. However, how mechanical signals are transduced in cells to regulate gene expression is largely unknown. We previously demonstrated that the gene encoding h2-calponin (Cnn2) is sensitively regulated by mechanical tension. In the present study, mouse genomic DNA containing the Cnn2 promoter was cloned, and a nested set of 5' truncations was studied. Transcriptional activity of the Cnn2 promoter-reporter constructs was examined in transfected NIH/3T3, HEK293, and C2C12 cells for their responses to the stiffness of culture substrate. The results showed significant transcriptional activities of the -1.00- and -1.24-kb promoter constructs, whereas the -0.61-kb construct was inactive. The -1.38-, -1.57-, and -2.12-kb constructs showed higher transcriptional activity, whereas only the -1.57- and -2.12-kb constructs exhibited repression of expression when the host cells were cultured on low stiffness substrate. Internal deletion of the segment between -1.57 and -1.38 kb in the -2.12-kb promoter construct abolished the low substrate stiffness-induced repression. Site-specific deletion or mutation of an HES-1 transcription factor binding site in this region also abolished this repression effect. The level of HES-1 increased in cells cultured under a low tension condition, corresponding to the down-regulation of h2-calponin. h2-Calponin gene expression is further affected by the treatment of cells with Notch inhibitor and activator, suggesting an upstream signaling mechanism.

Objective A nonapoptotic role of Fas signaling has been implicated in the regulation of inflammation and innate immunity. This study was undertaken to elucidate the contribution of Fas signaling in macrophages to the development of arthritis. Methods K/BxN serum–transfer arthritis was induced in a mouse line in which Fas was conditionally deleted in the myeloid lineage (CreLysMFasflox/flox mice). The arthritis was assessed clinically and histologically. Expression of interleukin-1β (IL-1β), CXCL5, IL-10, IL-6, and gp96 was determined by enzyme-linked immunosorbent assay. Bone marrow–derived macrophages were activated with IL-1β and gp96. Cell phenotype and apoptosis were analyzed by flow cytometry. Results Arthritis onset in CreLysMFasflox/flox mice was comparable with that observed in control mice; however, resolution was accelerated during the chronic phase. The attenuated arthritis was associated with reduced articular expression of the endogenous Toll-like receptor 2 (TLR-2) ligand gp96 and the neutrophil chemotactic chemokine CXCL5, and enhanced expression of IL-10. Activation with IL-1β or gp96 induced increased IL-10 expression in Fas-deficient murine macrophages compared with control macrophages. IL-10 suppressed IL-6 and CXCL5 expression induced by IL-1β plus gp96. IL-1β–mediated activation of ERK, which regulates IL-10 expression, was increased in Fas-deficient mouse macrophages. Conclusion Taken together, our findings indicate that impaired Fas signaling results in enhanced expression of antiinflammatory IL-10 and reduced expression of gp96, and these effects are associated with accelerated resolution of inflammation during the chronic phase of arthritis. These observations suggest that strategies to reduce endogenous TLR ligands and increase IL-10 may be beneficial in the treatment of rheumatoid arthritis.

TLR2 signaling modulates K/BxN serum transfer arthritis by enhancing the expression of immune complex-induced IL-10. RA is a chronic inflammatory disease characterized by the persistent expression of inflammatory cytokines from macrophages, which may be mediated, in part, through TLR2 signaling. Earlier studies demonstrate a role for TLR2 signaling in dampening the arthritis in IL-1Ra−/− mice, which was mediated through T cells. This study was performed to determine whether TLR2 signaling plays a role in the pathogenesis of T cell-independent arthritis triggered by transferring serum from K/BxN mice. We documented more severe arthritis in Tlr2−/− mice compared with WT controls. The Tlr2−/− mice also demonstrated increased inflammation, erosion, pannus formation, and osteoclastogenesis, as well as increased IL-1β and decreased IL-10 within the joints. In vitro bone marrow-differentiated macrophages expressed comparable levels of activating and inhibitory FcγRs, however when stimulated with immune complexes, the Tlr2−/− macrophages expressed decreased IL-10 and reduced activation of Akt and ERK. Our findings indicate that Tlr2−/− promotes the effector phase of arthritis through decreased IL-10 by macrophages, which is important, not only as an anti-inflammatory cytokine but also in restraining the differentiation and activation of osteoclasts.

The reduction of synovial tissue macrophages is a reliable biomarker for clinical improvement in patients with rheumatoid arthritis (RA), and macrophages are reduced in synovial tissue shortly after initiation of TNF inhibitors. The mechanism for this initial response is unclear. These studies were performed to identify the mechanisms responsible for the initial reduction of macrophages following TNF inhibition, positing that efflux to draining lymph nodes was involved. RA synovial tissue and synovial fluid macrophages expressed CCR7, which was increased in control macrophages following incubation with TNF-α. Human TNF transgenic (hTNF-Tg) mice were treated with infliximab after development of arthritis. Ankles were harvested and examined by histology, immunohistochemistry, quantitative RT-PCR, ELISA, and flow cytometry. hTNF-Tg mice treated with infliximab demonstrated significant clinical and histologic improvement 3 d after the initiation of therapy, at which time Ly6C+ macrophages were significantly reduced in the ankles. However, no evidence was identified to support a role of macrophage efflux to draining lymph nodes following treatment with infliximab. In contrast, apoptosis of Ly6C+ macrophages in the ankles and popliteal lymph nodes, decreased migration of monocytes into the ankles, and a reduction of CCL2 were identified following the initiation of infliximab. These observations demonstrate that Ly6C+ macrophage apoptosis and decreased ingress of circulating monocytes into the joint are responsible for the initial reduction of macrophages following infliximab treatment in hTNF-Tg mice.

The 96-kDa glycoprotein (gp96) is an endoplasmic reticulum (ER) resident molecular chaperone. Under physiologic conditions, gp96 facilitates the transport of toll-like receptors (TLRs) to cell or endosomal membranes. Under pathologic circumstances such as rheumatoid arthritis, gp96 translocates to the cell surface and extracellular space, serving as an endogenous danger signal promoting TLR signaling. Macrophages play a central role in regulating innate and adaptive immunity, and are the major source of proinflammatory cytokines and chemokines in rheumatoid arthritis (RA). Macrophage numbers in the sublining of RA synovial tissue correlate with clinical response. This review focuses on the recent findings that implicate gp96 induced macrophage activation mediated through TLR signaling in the pathogenesis of RA and provides insights concerning the targeting gp96 and the TLR signaling pathway as therapeutic approaches for patients with RA and possibly other chronic inflammatory conditions.

Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP is necessary for the differentiation and/or survival of macrophages. We also showed that FLIP is highly expressed in RA synovial macrophages. This study was undertaken to determine if a reduction in FLIP in mouse macrophages reduces synovial tissue macrophages and ameliorates serum-transfer arthritis.Mice with Flip deleted in myeloid cells (FlipIn contrast to expectations, FlipOur observations suggest that reducing FLIP in macrophages by increasing the number of antiinflammatory macrophages may be an effective therapeutic approach to suppress inflammation, depending on the disease stage.

FLIP is a well-established suppressor of death receptor-mediated apoptosis. To define its essential in vivo role in myeloid cells, we generated and characterized mice with Flip conditionally deleted in the myeloid lineage. Myeloid specific Flip-deficient mice exhibited growth retardation, premature death, and splenomegaly with altered architecture and extramedullary hematopoiesis. They also displayed a dramatic increase of circulating neutrophils and multiorgan neutrophil infiltration. In contrast, although circulating inflammatory monocytes were also significantly increased, macrophages in the spleen, lymph nodes, and the peritoneal cavity were reduced. In ex vivo cultures, bone marrow progenitor cells failed to differentiate into macrophages when Flip was deleted. Mixed bone marrow chimera experiments using cells from Flip-deficient and wild-type mice did not demonstrate an inflammatory phenotype. These observations demonstrate that FLIP is necessary for macrophage differentiation and the homeostatic regulation of granulopoiesis.

Abbreviations: –/–=deficient, ApoE=apolipoprotein E, CCP=cyclic-citrullinated peptide, DAMP=damage-associated molecular pattern, DC= dendritic cell, dsDNA=double-stranded DNA, ECM=extracellular matrix, EDA=extra domain A, gp96=96-kDa heat shock glycoprotein, HMGB1= high mobility group box chromosomal protein 1, HSP=heat shock protein, IKK=IκB kinase, IL-1Ra=IL-1R antagonist, IRAK=IL-1R-associated kinase, IVD macrophage=in vitro differentiated macrophage, LDL=low-density lipoprotein, LDLr=LDLR, Mal=MyD88 adaptor-like protein, MMP=matrix metalloproteinase, MyD88s=MyD88short, NZB=New Zealand Black, oxLDL=oxidized low-density lipoprotein, Pam3CSK4=palmitoyl-3-cysteine-serine-lysine-4, PAMP=pathogen-associated molecular pattern, pDC=plasmacytoid DC, PRR=pattern recognition receptor, RA=rheumatoid arthritis, RAGE=receptor for advanced glycation end-products, RNP=ribonucleoprotein, RP105=radioprotective 105, SCW=streptococcal cell wall, SLE=systemic lupus erythematosus, SRA=scavenger receptor A, TIR=Toll/IL-1R, TIRAP=Toll/IL-1R domain-containing adaptor protein, TRAF6=TNFR-associated factor 6, TRAM=TIR domain-containing adaptor-inducing IFN-α-related adaptor molecule, TRIF=Toll/IL-1R domain-containing adaptor-inducing IFN-α

Macrophages are important mediators of chronic inflammation and are prominent in the synovial lining and sublining of patients with rheumatoid arthritis (RA). Recently, we demonstrated increased TLR2 and TLR4 expression and increased response to microbial TLR2 and TLR4 ligands in macrophages from the joints of RA. The current study characterized the expression of the 96-kDa heat shock glycoprotein (gp96) in the joints of RA and its role as an endogenous TLR ligand to promote innate immunity in RA. gp96 was increased in RA compared with osteoarthritis and arthritis-free control synovial tissues. The expression of gp96 strongly correlated with inflammation and synovial lining thickness. gp96 was increased in synovial fluid from the joints of RA compared with disease controls. Recombinant gp96 was a potent activator of macrophages and the activation was mediated primarily through TLR2 signaling. The cellular response to gp96 was significantly stronger with RA synovial macrophages compared with peripheral blood monocytes from RA or healthy controls. The transcription of TLR2, TNF-α, and IL-8, but not TLR4, was significantly induced by gp96, and the induction was significantly greater in purified RA synovial macrophages. The expression of TLR2, but not TLR4, on synovial fluid macrophages strongly correlated with the level of gp96 in the synovial fluid. The present study documents the potential role of gp96 as an endogenous TLR2 ligand in RA and provides insight into the mechanism by which gp96 promotes the chronic inflammation of RA, identifying gp96 as a potential new therapeutic target.

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neoR cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neoR cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H+ pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.

Objective Macrophages are the major source of inflammation mediators that are important in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to analyze macrophages obtained from the joints of RA patients in order to characterize the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and the responses to TLR ligation. Methods Cells were isolated from the synovial fluid (SF) of RA patients or patients with other forms of inflammatory arthritis. Cell surface TLR-2 and TLR-4 expression and intracellular tumor necrosis factor α (TNFα) and interleukin-8 (IL-8) expression by CD14+ macrophages were determined by flow cytometry. Peptidoglycan (PG) and lipopolysaccharide (LPS) were used as ligands for TLR-2 and TLR-4, respectively. Results The expression of TLR-2 and TLR-4 was increased on CD14+ macrophages from the joints of RA patients compared with that on control in vitro–differentiated macrophages or control peripheral blood monocytes. Neither TLR-2 expression nor TLR-4 expression differed between RA and other forms of inflammatory arthritis. However, PG- and LPS-induced TNFα expression and IL-8 expression were greater with RA SF macrophages than with those obtained from the joints of patients with other forms of inflammatory arthritis or with control macrophages. PG-induced TNFα expression and IL-8 expression were highly correlated with TLR-2 expression in normal macrophages, but not with that in macrophages obtained from joints of RA patients or patients with other forms of inflammatory arthritis. Conclusion TLR-2 and TLR-4 ligation resulted in increased activation of RA synovial macrophages compared with those from patients with other forms of inflammatory arthritis or compared with control macrophages. Factors other than the level of TLR-2 and TLR-4 expression contributed to the increased activation of RA SF macrophages. These observations support the notion of a potential role for activation through TLR-2 and TLR-4 in the inflammation and joint destruction of RA.

Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag−/− mice. The CD8α+ DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4+ T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4+ T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α+ DCs and other cells of the immune system., Dendritic cells are critical for initiation of immune responses and for induction of tolerance. Here the authors show that deletion of survival factor c-flip in CD11c-expressing cells subset perturbs CD8a+ dendritic cell, NK and macrophage pools, and leads to development of autoimmune arthritis.

Receptor-interacting protein (RIP) has been implicated in the induction of death receptor-mediated, nonapoptotic cell death. However, the mechanisms remain to be elucidated. Here we show that tumor necrosis factor alpha induced RIP-dependent inhibition of adenine nucleotide translocase (ANT)-conducted transport of ADP into mitochondria, which resulted in reduced ATP and necrotic cell death. The inhibition of ADP/ATP exchange coincided with the loss of interaction between ANT and cyclophilin D and the inability of ANT to adopt the cytosolic conformational state, which prevented cytochrome c release. Neither overexpression of Bcl-xL nor inhibition of reactive oxygen species prevented necrosis. In contrast, the ectopic expression of ANT or cyclophilin D was effective at preventing cell death. These observations demonstrate a novel mechanism initiated through death receptor ligation and mediated by RIP that results in the suppression of ANT activity and necrosis.

Besides the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an N-terminal extension that contains phosphorylation sites for protein kinase A under beta-adrenergic regulation. A restricted cleavage of this N-terminal regulatory domain occurs in normal cardiac muscle and is up-regulated during hemodynamic adaptation (Z.-B. Yu, L.-F. Zhang, and J.-P. Jin (2001) J. Biol. Chem. 276, 15753-15760). In the present study, we developed transgenic mice overexpressing the N-terminal truncated cTnI (cTnI-ND) in the heart to examine its biochemical and physiological significance. Ca(2+)-activated actomyosin ATPase activity showed that cTnI-ND myofibrils had lower affinity for Ca(2+) than controls, similar to the effect of isoproterenol treatment. In vivo and isolated working heart experiments revealed that cTnI-ND hearts had a significantly faster rate of relaxation and lower left ventricular end diastolic pressure compared with controls. The higher baseline relaxation rate of cTnI-ND hearts was at a level similar to that of wild type mouse hearts under beta-adrenergic stimulation. The decrease in cardiac output due to lowered preload was significantly smaller for cTnI-ND hearts compared with controls. These findings indicate that removal of the N-terminal extension of cTnI via restricted proteolysis enhances cardiac function by increasing the rate of myocardial relaxation and lowering left ventricular end diastolic pressure to facilitate ventricular filling, thus resulting in better utilization of the Frank-Starling mechanism.

In vertebrates, three troponin T (TnT) genes, cardiac TnT (cTnT), skeletal muscle fast-twitch TnT (fTnT), and slow-twitch TnT (sTnT), have evolved for the regulation of striated muscle contraction. To understand the mechanism for muscle fiber-specific expression of the TnT genes, we compared their expression patterns during mouse development. Our data revealed that the TnT expression in the developing embryo was not as restricted as that in the adult. In addition to a strong expression in the developing heart beginning at day 7.5 p.c (postcoitum), the cTnT transcript was detected at later stages in some skeletal muscles, where beginning at day 11.75 p.c. the fTnT and sTnT genes were also expressed. Only sTnT but not fTnT was found transiently in the developing heart. At day 13.5 p.c., expressions of all three genes were detected in the developing tongue and this co-expression continued to day 16.5 p.c. with the fTnT isoform being predominant. At this stage, overlapping and distinct expression patterns of both sTnT and fTnT genes were also evident in many developing skeletal muscles. These data suggest that different muscles during development undergo a complex change in TnT isoforms resulting in different contractile properties. Unexpectedly, the cTnT transcript was persistently found in the developing bladder, where presumably smooth muscle is present. In transgenic mice, expression of a LacZ gene driven by a rat cTnT promoter (−497 to +192 bp) was very similar to that of the endogenous cTnT gene, suggesting that this promoter contained regulatory elements sufficient for the control of tissue-specific cTnT expression during development. Anat Rec 263:72–84, 2001. © 2001 Wiley-Liss, Inc.

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.

Ca(2+)-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca(2+) receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca(2+) signals into allosteric conformational changes of contractile proteins. While these Ca(2+) receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca(2+) regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca(2+) regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca(2+) receptor, troponin C, to the actin-myosin contractile machinery. This hypothesis views the three troponin subunits as two structure-function domains: the Ca(2+) receptor and the signal transducing adapter. This model may help to further our understanding of the Ca(2+) regulation of muscle contraction and the structure-function relationship of other potential adapter proteins which are converged to constitute the Ca(2+) signal transduction pathways governing nonmuscle cell motility.

1. To investigate the functional significance of different troponin T (TnT) isoforms in the Ca2+ activation of muscle contraction, transgenic mice have been constructed with a chicken fast skeletal muscle TnT transgene driven by a cardiac alpha-myosin heavy chain gene promoter. 2. Cardiac muscle-specific expression of the fast skeletal muscle TnT has been obtained with significant myofibril incorporation. Expression of the endogenous cardiac muscle thin filament regulatory proteins, such as troponin I and tropomyosin, was not altered in the transgenic mouse heart, providing an authentic system for the functional characterization of TnT isoforms. 3. Cardiac muscle contractility was analysed for the force vs. Ca2+ relationship in skinned ventricular trabeculae of transgenic mice in comparison with wild-type litter-mates. The results showed unchanged pCa50 values (5.1 +/- 0.04 and 5.1 +/- 0.1, respectively) but significantly steeper slopes (the Hill coefficient was 2.0 +/- 0.2 vs. 1.0 +/- 0.2, P0.05). 4. The results demonstrate that the structural and functional variation of different TnT isoforms may contribute to the difference in responsiveness and overall cooperativity of the thin filament-based Ca2+ regulation between cardiac and skeletal muscles.

Glutamic acid decarboxylase (GAD) is a pancreatic β cell autoantigen in humans and nonobese diabetic (NOD) mice. β Cell–specific suppression of GAD expression in two lines of antisense GAD transgenic NOD mice prevented autoimmune diabetes, whereas persistent GAD expression in the β cells in the other four lines of antisense GAD transgenic NOD mice resulted in diabetes, similar to that seen in transgene-negative NOD mice. Complete suppression of β cell GAD expression blocked the generation of diabetogenic T cells and protected islet grafts from autoimmune injury. Thus, β cell–specific GAD expression is required for the development of autoimmune diabetes in NOD mice, and modulation of GAD might, therefore, have therapeutic value in type 1 diabetes.

A nonapoptotic role of Fas signaling has been implicated in the regulation of inflammation and innate immunity. This study was undertaken to elucidate the contribution of Fas signaling in macrophages to the development of arthritis.K/BxN serum-transfer arthritis was induced in a mouse line in which Fas was conditionally deleted in the myeloid lineage (Cre(LysM) Fas(flox/flox) mice). The arthritis was assessed clinically and histologically. Expression of interleukin-1β (IL-1β), CXCL5, IL-10, IL-6, and gp96 was determined by enzyme-linked immunosorbent assay. Bone marrow-derived macrophages were activated with IL-1β and gp96. Cell phenotype and apoptosis were analyzed by flow cytometry.Arthritis onset in Cre(LysM) Fas(flox/flox) mice was comparable with that observed in control mice; however, resolution was accelerated during the chronic phase. The attenuated arthritis was associated with reduced articular expression of the endogenous Toll-like receptor 2 (TLR-2) ligand gp96 and the neutrophil chemotactic chemokine CXCL5, and enhanced expression of IL-10. Activation with IL-1β or gp96 induced increased IL-10 expression in Fas-deficient murine macrophages compared with control macrophages. IL-10 suppressed IL-6 and CXCL5 expression induced by IL-1β plus gp96. IL-1β-mediated activation of ERK, which regulates IL-10 expression, was increased in Fas-deficient mouse macrophages.Taken together, our findings indicate that impaired Fas signaling results in enhanced expression of antiinflammatory IL-10 and reduced expression of gp96, and these effects are associated with accelerated resolution of inflammation during the chronic phase of arthritis. These observations suggest that strategies to reduce endogenous TLR ligands and increase IL-10 may be beneficial in the treatment of rheumatoid arthritis.

Analysis of deduced protein sequence and structural motifs of ~5500 residues of human fetal skeletal muscle nebulin reveals the design principles of this giant multifunctional protein in the sarcomere. The bulk of the sequence is constructed of ~150 tandem copies of ~35-residue modules that can be classified into seven types. The majority of these modules form 20 super-repeats, with each super-repeat containing a 7-module set (one of each type in the same order). These super-repeats are further divided into eight segments: with six segments containing adjacent, highly homologous super-repeats, one single repeat segment consisting of 8 nebulin modules of the same type, and a non-repeat segment terminating with a SH3 domain at the C terminus. The interactions of actin, tropomyosin, troponin, and calmodulin with nebulin fragments consisting of either repeating modules or the SH3 domain support its role as a giant actin-binding cofilament of the composite thin filament. Such affinity profiles also suggest that nebulin may bind to tropomyosin and troponin to form a composite calcium-linked regulatory complex on the thin filament. The modular construction, super-repeat structure, and segmental organization of nebulin sequence appear to encode thin filament length, periodicity, insertion, and sarcomere proportion in the resting muscle.

Neuronal cdc2-like kinase, nclk, is a heterodimer of cyclin dependent protein kinase 5, cdk5, and a 25 kDa subunit derived from a novel, neuron-specific, 35 kDa protein: p35. The characterization and regulation of nclk will be summarized in this minireview. The activity of nclk appears to be governed by highly complex regulatory mechanisms including protein-protein interaction, protein phosphorylation and isoforms. The histone H1 kinase activity of nclk is absolutely dependent of the interaction between the 25 kDa subunit and the catalytic subunit, cdk5. In addition, nclk interacts with other cellular proteins to form macromolecular complexes. The kinase activity of nclk is inhibited in vitro by the phosphorylation reactions of a weel-like protein tyrosine kinase and a protein serine/threonine kinase from bovine thymus. Northern blot analysis has revealed the existence of two populations of p35 mRNA of 2 and 4 kb. A novel cDNA encoding a p35 homologous protein has been obtained from a human hippocampus library.

We have identified and purified from bovine brain a novel protein kinase which catalyzes in vitro phosphorylation of neurofilament proteins NF-H and NF-M and tau proteins at sites implicating the enzyme in the regulation of neurocytoskeleton dynamics and in Alzheimer pathology. The protein kinase displays a phosphorylation site specificity similar or identical to the cell cycle regulatory kinase, cdc2 kinase. The purified kinase is a heterodimer of a cdc2-like catalytic subunit, called cdk5, and a 25 kDa regulatory subunit. The regulatory subunit is essential for kinase activity, and it is derived from a 35 kDa protein, p35 by proteolysis. Northern blot analysis of tissue distribution indicates that cdk5 is widely distributed but especially rich in brain, whereas p35 expression is only found in brain. The protein kinase is therefore termed neuronal cdc2-like kinase. The neuron-specificity of the enzyme appears to be conferred by the regulatory subunit. During cell division, cdc2 kinase is regulated by complex phosphorylation mechanisms involving a network of specific protein kinases. Some of these kinases or their homologs have been found in mammalian brains and they may be involved in the regulation of neuronal cdc2-like kinase.

Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit which is derived from a brain-specific 35-kDa novel protein, p35 (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R. J., Aebersold, R., Hunt, T., and Wang, J. H. (1994) Nature 371, 423-426). Three truncated forms of p35 including the one corresponding to the 25-kDa subunit of the kinase have been expressed in Escherichia coli and shown to activate a bacteria-expressed Cdk5 with equal efficacy. The shortest truncated form of p35, p21, spanning amino acid residues 88 to 291, has been used to reconstitute active Cdk5 kinase and to characterize the activation reaction. The purified kinase displays similar specific enzyme activity and similar phosphorylation site specificity as the neuronal Cdc2-like kinase purified from bovine brain. Bovine brain extract contains Cdk5 uncomplexed with p35 or p25 which has also been found to be activated by p21 or p25. The results substantiate the previous suggestion that p35 is a specific Cdk5 activator. Several observations suggest that, unlike other well characterized Cdc2-like kinases whose activities depend on the phosphorylation of the catalytic subunits at a specific site by a distinct kinase, the reconstituted Cdk5/p21 does not depend on the phosphorylation of Cdk5 for activity. The reconstitution of the highly active Cdk5 kinase was achieved without requiring any other kinase in the reconstitution reaction. The possibility of autophosphorylation of Cdk5 on the putative activation site has been ruled out as no phosphorylation occurred on Cdk5 during the enzyme reaction. The rate and extent of the kinase reconstitution were not significantly affected by Mg2+ ATP.

This paper proposes an efficient downlink resource allocation scheme for Orthogonal Frequency Division Multiple Access based WiMAX systems using the Adaptive Modulation and Coding mode operation. Given a set of user requests, the proposed algorithm determines the modulation scheme and allocates a two-dimensional (time and frequency) burst to each user. It is known that finding the optimal solution for such a resource allocation problem is NP-complete. To reduce the computation complexity, this paper proposes a heuristic solution. Compared with the Efficient Downlink Bandwidth Allocation (EDBA) algorithm [6], the proposed scheme achieves higher channel utilization and system throughput with similar execution time. Simulation results show that the average channel utilization of the proposed scheme is about 75∼85% and that of EDBA is about 60∼70%.

Objective The mechanisms contributing to the persistent activation of macrophages in rheumatoid arthritis (RA) are not fully understood. Some studies suggest that endogenous toll-like receptor (TLR) ligands promote the chronic inflammation observed in RA. The objective of this study was to identify endogenous TLR ligands expressed in RA synovial tissue (ST) based on their ability to bind the extracellular domains of TLR2 or TLR4. Methods A yeast two-hybrid cDNA library was constructed from ST obtained by arthroscopy from patients with RA and screened using the extracellular domains of TLR2 and TLR4 as the bait. Interactions between TLRs and Snapin were demonstrated by reciprocal co-immunoprecipitation. ST was examined by histology, and single- and two-colour immunohistochemistry and quantitative reverse transcriptase PCR. Snapin (SNAP – associated protein) expression in macrophages was examined by Western Blot analysis and confocal microscopy. The ability of Snapin to activate through TLR2 was examined. Results Employing a yeast two-hybrid system, Snapin was the most frequently identified molecule that interacted with TLR2. These results were confirmed by pull-down of in vitro-expressed Snapin together with TLR2. By immunohistochemistry and quantitative reverse transcriptase PCR, Snapin was highly expressed in RA ST, and it was readily detected in macrophages, where it co-localised in the late endosomes. ST Snapin expression correlated with inflammation and was not disease specific. Finally, Snapin was capable of activating through TLR2. Conclusion These observations identify Snapin as a novel endogenous TLR2 ligand in RA, and thus support a role for persistent TLR2 signalling in the pathogenesis of RA.

Expression screening in Xenopus oocytes was used to isolate a cDNA from rat jejunal epithelium encoding a Na(+)-dependent nucleoside transport protein (named cNT1). The cDNA sequence of cNT1 predicts a protein of 648 amino acids (relative molecular mass 71,000) with 14 potential transmembrane domains. Data base searches indicate significant sequence similarity to the NUPC proton/nucleoside symporter of Escherichia coli. There is no sequence similarity between cNT1 and proteins of mammalian origin. Functionally, cNT1 exhibited the transport characteristics of the nucleoside transport system cit (selective for pyrimidine nucleosides and adenosine) and accepted both 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) as permeants (Km = 0.49 and 0.51 mM, respectively). The demonstration of transport of AZT by cNT1 expressed in Xenopus oocytes provides the first direct evidence that AZT enters cells by transporter-mediated processes, as well as by passive diffusion. Consistent with the tissue distribution of system cit transport activity, transcripts for cNT1 were detected in kidney as well as jejunum. cNT1 therefore belongs to a potential new gene family and may be involved in the intestinal absorption and renal handling of pyrimidine nucleoside analogs used to treat acquired immunodeficiency syndrome (AIDS).

Hepatocarcinogenesis in rodents induced by nongenotoxic peroxisome proliferators is postulated to be a receptor-mediated process. The peroxisome proliferator-activated receptors (PPAR) are members of the steroid hormone receptor superfamily, which participate in ligand-dependent transcriptional activation of peroxisomal fatty acid beta oxidation enzyme system genes in liver parenchymal cells of rats and mice. In order to study the tissue distribution and cellular localization of PPAR, we raised polyclonal antibodies against PPAR using a recombinant rat PPAR (rPPAR) expressed as a glutathione-S-transferase-rPPAR fusion protein. On immunoblot analysis the antibodies specifically recognized a 55 kDa PPAR protein in rat, mouse and human liver homogenates. Immunoblotting also showed that in the mouse and rat, PPAR is expressed in liver, kidney and heart, and only weakly in brain and testis. Immunohistochemical localization in the rat and mouse revealed that PPAR is highly expressed in perivenular (i.e., those surrounding hepatic vein) hepatocytes and very weakly in the cytoplasm of remaining hepatocytes. In the kidney, PPAR was visualized predominantly in the p(3) segments of proximal convoluted tubular epithelium. CV-1 cells transiently transfected with rPPAR cDNA construct showed predominant cytoplasmic fluorescence; treatment of these cells with ciprofibrate, a peroxisome proliferator, resulted in the nuclear translocation of PPAR signal.

Nebulin, a giant myofibrillar protein with size variants from 700 to 900 kDa in various skeletal muscles, has been proposed to constitute a set of inextensible filaments anchored at the Z-line and coextensive with actin filaments. To elucidate the architectural organization of this fourth set of myofilaments in the skeletal muscle sarcomere, we performed immunoelectron microscopic localization of epitope profiles of a number of site-specific monoclonal antibodies against cloned human nebulin fragments of known sequence loci. Monoclonal antibody N113, which is directed to fragment ND8 at approximately 300 residues away from the C-terminus, labelled the edges of Z-lines in both human quadriceps muscle and rabbit psoas muscle. Monoclonal antibody N101, which is directed to fragment NB5 near the N-terminal side, is localized to a single locus at 0.89 micron from the Z-line in human quadriceps muscle and 0.80 micron from the Z-line in rabbit psoas muscle. Additionally, monoclonal antibody N109, which is directed to fragment NA3 on the carboxy side of the adjacent fragment NB5, is localized at 0.76 micron away from the Z-line in rabbit psoas muscle. This one-to-one correspondence between epitope loci and sequence loci demonstrates that a single nebulin polypeptide spans the length of the thin filament with its C-terminus anchored at the Z-line. The epitope spacings of site-specific antibodies are consistent with the notion that the nebulin filament is uniform in mass density along its length.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated stage VI oocytes from Xenopus laevis expressed uridine transport activity after microinjection of mRNA from rat jejunum. Uridine uptake during 30 min (10 microM, 20 degrees C) by mRNA-injected oocytes reached 2.5 pmol/oocyte, compared with endogenous uptake by water-injected oocytes of about 0.05 pmol/oocyte. The expressed transport activity was 96% Na(+)-dependent, saturable (apparent Km = 15 microM) and inhibited by phloridzin (IC50 = 100 microM). Nucleoside inhibition studies resolved the expressed transport activity into two components: 1) a novel Na(+)-dependent system of broad purine and pyrimidine specificity that was inhibited by low concentrations of guanosine, inosine, adenosine, uridine, thymidine, and cytidine and 2) a Na(+)-dependent system of narrower specificity that was inhibited by low concentrations of guanosine, inosine, adenosine, and uridine and by high concentrations of thymidine and cytidine. The characteristics of the latter system are consistent with those of the Na(+)-dependent nucleoside transport system N1 (cif), previously identified in a number of cell types and tissues, including intestinal epithelia and cultured cells of intestinal origin. The broad specificity system, which was also detected in mRNA-injected oocytes using thymidine as permeant, has been given the provisional designation N3 to distinguish it from the previously described N1 (purine-selective) and N2 (pyrimidine-selective) Na(+)-linked nucleoside transporters. Rat jejunal transporters N1 and N3 were both expressed maximally by the same mRNA size fraction (1.6-3.0 kb, peak 2.3 kb).

Rheumatoid arthritis (RA) is a chronic inflammatory disease that is mediated, in part, by proinflammatory factors produced by RA synovial tissue (ST) fibroblasts and macrophages, resulting in monocyte migration from the blood to the ST. To characterize the potential role of IL-17 in monocyte migration, RA synovial fibroblasts and macrophages were activated with IL-17 and examined for the expression of monocyte chemokines. The two potentially important monocyte chemoattractants identified were CCL20/MIP-3α and CCL2/MCP-1, which were significantly induced in RA synovial fibroblasts and macrophages. However, in vivo, only CCL2/MCP-1 was detectable following adenovirus IL-17 injection. We found that IL-17 induction of CCL2/MCP-1 was mediated by the PI3K, ERK, and JNK pathways in RA ST fibroblasts and by the PI3K and ERK pathways in macrophages. Further, we show that neutralization of CCL2/MCP-1 significantly reduced IL-17–mediated monocyte recruitment into the peritoneal cavity. We demonstrate that local expression of IL-17 in ankle joints was associated with significantly increased monocyte migration and CCL2/MCP-1 levels. Interestingly, we show that RA synovial fluids immunoneutralized for IL-17 and CCL2/MCP-1 have similar monocyte chemotaxis activity as those immunoneutralized for each factor alone. In short, CCL2/MCP-1 produced from cell types present in the RA joint, as well as in experimental arthritis, may be responsible, in part, for IL-17–induced monocyte migration; hence, these results suggest that CCL2/MCP-1 is a downstream target of IL-17 that may be important in RA.

We have previously demonstrated that rat cardiac troponin T (TnT) is expressed as two different isoforms during development, the larger, more acidic embryonic isoform and the smaller, more basic adult isoform, which appear to be generated from a common transcript of the cardiac TnT gene by alternative RNA splicing. In this study, Southern blot analysis confirmed the existence of a single copy of cardiac TnT gene in the rat genome. For investigation of the molecular mechanism of isoform switch and the control of this gene expression in myocardial development, several overlapping genomic clones were isolated from a rat genomic library. Complete nucleotide sequences were determined from these genomic clones and revealed a 19,186 base-pair DNA fragment containing 16 exons of rat cardiac TnT gene. Its DNA sequence and exon organization appeared to differ from that of the rat fast skeletal muscle TnT gene or chicken cardiac TnT gene. Comparison of genomic and cDNA clones also confirmed that the cardiac TnT isoform switching was due to the inclusion or exclusion of exon 4 during RNA processing. Sequence analysis allowed us to further identify the other alternatively spliced exon containing only nine nucleotides in size (exon 12). The inclusion and complete or partial exclusion of this exon may be responsible for generating three classes of mRNAs detected by our cDNA clones. The functional significance of this variation in TnT isoforms remained unknown, but its splicing pattern did not appear to link to the developmental changes. The 5' upstream structure was very similar to that in chicken cardiac TnT gene but differed from that in the rat fast skeletal muscle TnT gene, suggesting a similar regulatory mechanism for mammalian and avian cardiac TnT expression.

An increasing body of data supports the role of the innate immune system in the pathogenesis of rheumatoid arthritis (RA). Toll-like receptors (TLRs) are expressed by cells within the RA joint and a variety of endogenous TLR ligands are present within the inflamed joints of patients with RA. Further, a variety of animal models suggest that TLR signaling is important in the pathogenesis of disease. Overall, the data suggest that activation by endogenous TLR ligands may contribute to the persistent expression of pro-inflammatory cytokines by macrophages and the joint damage to cartilage and bone that occurs in RA. The data supports a potential role for suppression of TLR signaling as a novel therapeutic approach in patients with RA.

In contrast to skeletal muscles that simultaneously express multiple troponin T (TnT) isoforms, normal adult human cardiac muscle contains a single isoform of cardiac TnT. To understand the significance of myocardial TnT homogeneity, we examined the effect of TnT heterogeneity on heart function. Transgenic mouse hearts overexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT was investigated in vivo and ex vivo as an experimental system of concurrent presence of two classes of TnT in the adult cardiac muscle.This model of myocardial TnT heterogeneity produced pathogenic phenotypes: echocardiograph imaging detected age-progressive reductions of cardiac function; in vivo left ventricular pressure analysis showed decreased myocardial contractility; ex vivo analysis of isolated working heart preparations confirmed an intrinsic decrease of cardiac function in the absence of neurohumoral influence. The transgenic mice also showed chronic myocardial hypertrophy and degeneration. The dominantly negative effects of introducing a fast TnT into the cardiac thin filaments to produce two classes of Ca2+regulatory units in the adult myocardium suggest that TnT heterogeneity decreases contractile function by disrupting the synchronized action during ventricular contraction that is normally activated as an electrophysiological syncytium.

Calponins form an evolutionary highly conserved family of actin filament-associated proteins expressed in both smooth muscle and non-muscle cells. Whereas calponin-1 and calponin-2 have already been studied to some extent, little is known about the role of calponin-3 under physiological conditions due to the lack of an appropriate animal model. Here, we have used an unbiased screen to identify novel proteins implicated in signal transduction downstream of the precursor B cell receptor (pre-BCR) in B cells. We find that calponin-3 is expressed throughout early B cell development, localizes to the plasma membrane and is phosphorylated in a Syk-dependent manner, suggesting a putative role in pre-BCR signaling. To investigate this in vivo, we generated a floxed calponin-3-GFP knock-in mouse model that enables tracking of cells expressing calponin-3 from its endogenous promoter and allows its tissue-specific deletion. Using the knock-in allele as a reporter, we show that calponin-3 expression is initiated in early B cells and increases with their maturation, peaking in the periphery. Surprisingly, conditional deletion of the Cnn3 revealed no gross defects in B cell development despite this regulated expression pattern and the in vitro evidence, raising the question whether other components may compensate for its loss in lymphocytes. Together, our work identifies calponin-3 as a putative novel mediator downstream of the pre-BCR. Beyond B cells, the mouse model we generated will help to increase our understanding of calponin-3 in muscle and non-muscle cells under physiological conditions.

Objective Resistance to apoptosis may be an important mechanism contributing to the persistence of rheumatoid arthritis (RA). This study was undertaken to characterize the expression, regulation, and function of the antiapoptotic Bcl-2 family member Mcl-1 in macrophages isolated from the joints of patients with RA. Methods Mononuclear cells were isolated from the synovial fluid (SF) of patients with RA. Mcl-1 expression was documented by intracellular staining of CD14+ cells using flow cytometry, and by real-time polymerase chain reaction or immunoblot analysis of isolated macrophages. The expression of Mcl-1 was suppressed with small interfering RNA (siRNA) or chemical inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-1 and signal transducer and activator of transcription 3 (STAT-3) pathways. Apoptosis was defined by the loss of mitochondrial transmembrane potential and by DNA fragmentation. Results The expression of Mcl-1 was increased in CD14+ macrophages from the SF of patients with RA compared with normal in vitro–differentiated macrophages. Inhibition of the PI 3-kinase/Akt-1 or STAT-3 pathways significantly reduced the percentage of CD14+ cells within the SF and resulted in the reduction of Mcl-1 and the induction of apoptosis of synovial macrophages. Transfection of RA synovial macrophages with Mcl-1 siRNA resulted in apoptotic cell death. Conclusion Mcl-1 is critical for the survival of macrophages in the joints of patients with RA, and is therefore a potential therapeutic target in this disease.

A nonsense mutation at codon Glu180 in exon 11 of slow skeletal muscle troponin T (TnT) gene (TNNT1) causes an autosomal-recessive inherited nemaline myopathy. We previously reported the absence of intact or prematurely terminated slow TnT polypeptide in Amish nemaline myopathy (ANM) patient muscle. The present study further investigates the expression and fate of mutant slow TnT in muscle cells. Intact slow TnT mRNA was readily detected in patient muscle, indicating unaffected transcription and RNA splicing. Sequence of the cloned cDNAs revealed the single nucleotide mutation in two alternatively spliced isoforms of slow TnT mRNA. Mutant TNNT1 cDNA is translationally active in Escherichia coli and non-muscle eukaryotic cells, producing the expected truncated slow TnT protein. The mutant mRNA was expressed at significant levels in differentiated C2C12 myotubes, but unlike intact exogenous TnT, truncated slow TnT protein was not detected. Transfective expression in undifferentiated myoblasts produced slow TnT mRNA but not a detectable amount of truncated or intact slow TnT proteins, indicating a muscle cell-specific proteolysis of TnT when it is not integrated into myofilaments. The slow TnT-(1–179) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosin-binding sites. Our findings suggest that inefficient incorporation into myofilament is responsible for the instability of mutant slow TnT in ANM muscle. Rapid degradation of the truncated slow TnT protein, rather than instability of the nonsense mRNA, provides the protective mechanism against the potential dominant negative effect of the mutant TnT fragment.

Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3'-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p

A lethal form of nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), is linked to a nonsense mutation at codon Glu180 in the slow skeletal muscle troponin T (TnT) gene. We found that neither the intact nor the truncated slow TnT protein was present in the muscle of patients with ANM. The complete loss of slow TnT is consistent with the observed recessive pattern of inheritance of the disease and indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils. Expression of slow and fast isoforms of TnT is fiber-type specific. The lack of slow TnT results in selective atrophy of type 1 fibers. Slow TnT confers a higher Ca2+ sensitivity than does fast TnT in single fiber contractility assays. Despite the lack of slow TnT, individuals with ANM have normal muscle power at birth. The postnatal onset and infantile progression of ANM correspond to a down-regulation of cardiac and embryonic splice variants of fast TnT in normal developing human skeletal muscle, suggesting that the fetal TnT isoforms complement slow TnT. These results lay the foundation for understanding the molecular pathophysiology and the potential targeted therapy of ANM.

h2-Calponin is found in both smooth muscle and nonmuscle cells, and its function remains to be established. Western blots with specific monoclonal antibodies detected significant expression of h2-calponin in the growing embryonic stomach and urinary bladder and the early pregnant uterus. Although the expression of h1-calponin is upregulated in the stomach and bladder during postnatal development, the expression of h2-calponin is decreased to low levels in quiescent smooth muscle cells. To investigate a hypothesis that h2-calponin regulates the function of the actin cytoskeleton during cytokinesis, a smooth muscle-originated cell line (SM3) lacking calponin was transfected to express either sense or antisense h2-calponin cDNA and the effects on the rates of cell proliferation were examined. Both stable and transient sense cDNA-transfected cells had a significantly decreased proliferation rate compared with the antisense cDNA-transfected or nontransfected cells. Immunofluorescence microscopy showed that the force-expressed h2-calponin was associated with actin-tropomyosin microfilaments. The number of binuclear cells was significantly greater in the sense cDNA-transfected culture, in which h2-calponin was concentrated in a nuclear ring structure formed by actin filaments. The results suggest that h2-calponin may regulate cytokinesis by inhibiting the activity of the actin cytoskeleton.

Protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) and troponin T (cTnT) has been shown to diminish maximum activation of myofilaments. The functional role of cTnI phosphorylation has been investigated. However, the impact of cTnT phosphorylation on myofilament force is not well studied. We tested the effect of endogenous PKC activation on steady-state tension development and Ca2+sensitivity in skinned fiber bundles from transgenic (TG) mouse hearts expressing fast skeletal TnT (fsTnT), which naturally lacks the PKC sites present in cTnT. The 12- O-tetradecanoylphorbol 13-acetate (TPA) treatment induced a 29% (46.1 ± 2.5 vs. 33.4 ± 2.6 mN/mm2) reduction in maximum tension in the nontransgenic (NTG) preparations ( n = 7) and was inhibited with chelerythrine. However, TPA did not induce a change in the maximum tension in the TG preparations ( n = 11). TPA induced a small but significant ( P < 0.02) increase in Ca2+sensitivity (untreated pCa50= 5.63 ± 0.01 vs. treated pCa50= 5.72 ± 0.01) only in TG preparations. In TG preparations,32P incorporation was not evident in TnT and was also significantly diminished in cTnI, compared with NTG. Our data indicate that incorporation of fsTnT into the cardiac myofilament lattice blunts PKC-mediated depression of maximum tension. These data also suggest that cTnT may play an important role in amplifying the myofilament depression induced by PKC-mediated phosphorylation of cTnI.

Cultured airway smooth muscle cells subjected to cyclic deformational strain have increased cell content of myosin light chain kinase (MLCK) and myosin and increased formation of actin filaments. To determine how these changes may increase cell contractility, we measured isometric force production with changes in cytosolic calcium in individual permeabilized cells. The pCa for 50% maximal force production was 6.6 ± 0.4 in the strain cells compared with 5.9 ± 0.3 in control cells, signifying increased calcium sensitivity in strain cells. Maximal force production was also greater in strain cells (8.6 ± 2.9 vs. 5.7 ± 3.1 μN). The increased maximal force production in strain cells persisted after irreversible thiophosphorylation of myosin light chain, signifying that increased force could not be explained by differences in myosin light chain phosphorylation. Cells strained for brief periods sufficient to increase cytoskeletal organization but insufficient to increase contractile protein content also produced more force, suggesting that strain-induced cytoskeletal reorganization also increases force production.

Troponin T (TnT) is an essential element in the thin filament Ca2+-regulatory system controlling striated muscle contraction. Alternative RNA splicing generates developmental and muscle type-specific TnT isoforms differing in the hypervariable NH2-terminal region. Using avian fast skeletal muscle TnT containing a metal-binding segment, we have demonstrated a role of the NH2-terminal domain in modulating the conformation of TnT (Wang J and Jin JP. Biochemistry 37: 14519–14528, 1998). To further investigate the structure-function relationship of TnT, the present study constructed and characterized a recombinant protein in which the metal-binding peptide present in avian fast skeletal muscle TnT was fused to the NH2terminus of mouse slow skeletal muscle TnT. Metal ion or monoclonal antibody binding to the NH2-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by the altered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to the NH2-terminal extension had effects on the interaction of TnT with troponin I, troponin C, and most significantly, tropomyosin. The data indicate that the NH2-terminal Tx [4–7 repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slow skeletal muscle TnT.

We have cloned and sequenced full-length cDNAs encoding mouse slow skeletal muscle troponin T (sTnT). Alternative mRNA splicing-generated two high Mr isoforms and one low Mr sTnT isoform differing in the NH2-terminal primary structure have been identified by Western blotting, reverse transcription-polymerase chain reaction and cDNA cloning/expression analyses. Together with a 5′-alternative exon that was also found in human sTnT encoding an 11-amino-acid acidic segment, the results revealed a novel alternative splicing pathway to include or exclude a three-base segment to generate additional sTnT isoforms with NH2-terminal charge variations. Overriding the phylogenetic divergence, primary structure of sTnT is better conserved between mammalian and avian species than that of cardiac, fast and skeletal muscle TnTs from one species. Western blots demonstrate four expression patterns of sTnT during postnatal skeletal muscle development: (1) a decrease to a non-detectable level in mouse masseter, (2) an increase to become the sole TnT in sheep masseter, (3) an increase of the total level as well as the proportion of the low Mr isoform in sheep diaphragm and, (4) no significant change in total level or high/low Mr isoform ratio in sheep gastrocnemius. The highly conserved primary structure and fiber type-specific and developmentally regulated expression of sTnT indicate a physiological importance of this under-studied member of the TnT gene family.

Introduction: Our objectives were to examine mononuclear cell gene expression profiles in patients with systemic lupus erythematosus (SLE) and healthy controls and to compare subsets with and without atherosclerosis to determine which genes’ expression is related to atherosclerosis in SLE. Methods: Monocytes were obtained from 20 patients with SLE and 16 healthy controls and were in vitro-differentiated into macrophages. Subjects also underwent laboratory and imaging studies to evaluate for subclinical atherosclerosis. Whole-genome RNA expression microarray was performed, and gene expression was examined. Results: Gene expression profiling was used to identify gene signatures that differentiated patients from controls and individuals with and without atherosclerosis. In monocytes, 9 out of 20 patients with SLE had an interferon-inducible signature compared with 2 out of 16 controls. By looking at gene expression during monocyte-to-macrophage differentiation, we identified pathways which were differentially regulated between SLE and controls and identified signatures based on relevant intracellular signaling molecules which could differentiate SLE patients with atherosclerosis from controls. Among patients with SLE, we used a previously defined 344-gene atherosclerosis signature in monocyte-to-macrophage differentiation to identify patient subgroups with and without atherosclerosis. Interestingly, this signature further classified patients on the basis of the presence of SLE disease activity and cardiovascular risk factors. Conclusions: Many genes were differentially regulated during monocyte-to-macrophage differentiation in SLE patients compared with controls. The expression of these genes in mononuclear cells is important in the pathogenesis of SLE, and molecular profiling using gene expression can help stratify SLE patients who may be at risk for development of atherosclerosis.