Using a Xenopus expression-cloning screen, we have isolated Gremlin, a novel antagonist of bone morphogenetic protein (BMP) signaling that is expressed in the neural crest. Gremlin belongs to a novel gene family that includes the head-inducing factor Cerberus and the tumor suppressor DAN. We show that all family members are secreted proteins and that they act as BMP antagonists in embryonic explants. We also provide support for the model that Gremlin, Cerberus, and DAN block BMP signaling by binding BMPs, preventing them from interacting with their receptors. In addition, Cerberus alone blocks signaling by Activin- and Nodal-like members of the TGF beta superfamily. Therefore, we propose that Gremlin, Cerberus, and DAN control diverse processes in growth and development by selectively antagonizing the activities of different subsets of the TGF beta ligands.

Transforming growth factor-beta (TGF-beta) inhibits osteoblast differentiation through inhibition of the function of Runx2 (Cbfa1) by Smad3. The mechanism through which TGF-beta/Smad3 inhibits Runx2 function has not been characterized. We show that TGF-beta induces histone deacetylation, primarily of histone H4, at the osteocalcin promoter, which is repressed by TGF-beta, and that histone deacetylation is required for repression of Runx2 by TGF-beta. This repression occurs through the action of the class IIa histone deacetylases (HDAC)4 and 5, which are recruited through interaction with Smad3 to the Smad3/Runx2 complex at the Runx2-binding DNA sequence. Accordingly, HDAC4 or 5 is required for efficient TGF-beta-mediated inhibition of Runx2 function and is involved in osteoblast differentiation. Our results indicate that class IIa HDACs act as corepressors for TGF-beta/Smad3-mediated transcriptional repression of Runx2 function in differentiating osteoblasts and are cell-intrinsic regulators of osteoblast differentiation.

Mesenchymal stem (stromal) cells (MSCs) are rare, multipotent progenitor cells that can be isolated and expanded from bone marrow and other tissues. Strikingly, MSCs modulate the functions of immune cells, including T cells, B cells, natural killer cells, monocyte/macrophages, dendritic cells, and neutrophils. T cells, activated to perform a range of different effector functions, are the primary mediators of many autoimmune and inflammatory diseases as well as of transplant rejection and graft-versus-host disease. Well-defined T-cell effector phenotypes include the CD4+ (T helper cell) subsets Th1, Th2, and Th17 cells and cytotoxic T lymphocytes derived from antigen-specific activation of naïve CD8+ precursors. In addition, naturally occurring and induced regulatory T cells (Treg) represent CD4+ and CD8+ T-cell phenotypes that potently suppress effector T cells to prevent autoimmunity, maintain self-tolerance, and limit inflammatory tissue injury. Many immune-mediated diseases entail an imbalance between Treg and effector T cells of one or more phenotypes. MSCs broadly suppress T-cell activation and proliferation in vitro via a plethora of soluble and cell contact-dependent mediators. These mediators may act directly upon T cells or indirectly via modulation of antigen-presenting cells and other accessory cells. MSC administration has also been shown to be variably associated with beneficial effects in autoimmune and transplant models as well as in several human clinical trials. In a small number of studies, however, MSC administration has been found to aggravate T cell-mediated tissue injury. The multiple effects of MSCs on cellular immunity may reflect their diverse influences on the different T-cell effector subpopulations and their capacity to specifically protect or induce Treg populations. In this review, we focus on findings from the recent literature in which specific modulatory effects of MSCs on one or more individual effector T-cell subsets and Treg phenotypes have been examined in vitro, in relevant animal models of in vivo immunological disease, and in human subjects. We conclude that MSCs have the potential to directly or indirectly inhibit disease-associated Th1, Th2, and Th17 cells as well as cytotoxic T lymphocytes but that many key questions regarding the potency, specificity, mechanistic basis, and predictable therapeutic value of these modulatory effects remain unanswered.

Polyomavirus enhancer binding protein, PEBP2 (PEA2), is a heterodimer of two distinct subunits, alpha and beta, of which the former directly binds to DNA and the latter acts auxiliary to enhance the DNA binding. Recent cloning studies has revealed that the alpha subunit is homologous to the products of the Drosophila segmentation gene runt and the human AML1 gene, and that it functions as a major regulator for the T cell-specific gene expression. We have currently cloned cDNAs for the beta subunit. The isolated cDNAs contain three isoforms that are presumed to arise from alternative RNA splicing and encode polypeptides consisting of 187, 182, and 155 amino acids, respectively. These polypeptides neither show any significant homology with known other proteins including the alpha subunit nor have any known DNA-binding and dimerization domains. Thus, PEBP2, as the complex of these subunits, is thought to constitute an entirely novel category of heteromeric transcriptional regulator together with the Runt and AML1 proteins. Gel retardation assays of the cDNA-encoded proteins produced in an in vitro translation system or in Escherichia coli demonstrated that the larger two beta isoforms, but not the smallest one, can dimerize with the alpha subunit. Furthermore, this heterodimerization was shown to cause a marked increase in the intrinsic DNA binding affinity of the alpha subunit.

The introns separating the variable and constant regions of active immunoglobulin genes contain tissue-specific transcriptional enhancer elements, DNA segments which act in cis in an orientation- and distance-independent (up to a few kilobases (kb)) manner to enhance transcription initiation at adjacent promoters. The immunoglobulin heavy-chain enhancer is active only in lymphoid cells: in transfection assays it is capable of controlling in cis transcription from the simian virus 40 (SV40) T-antigen, rabbit beta-globin and immunoglobulin gene promoters up to at least 2 kb away. Genetic deletion analysis suggests that a region of as few as 140 base pairs (bp) is sufficient for the enhancement effect. These functional characteristics and DNA sequences are conserved between mouse and man. However, it is not known whether tissue-specific proteins bind to the enhancer. Proteins that interact with DNA at specific sequences can prevent or enhance the reactions of individual guanines or adenines with dimethyl sulphate (DMS), and this property has been used to display the DNA contacts of various regulatory proteins. Here we apply this DMS strategy in experiments involving single-copy genes within intact mammalian nuclei using genomic sequencing.

Sewage, a complex mixture of organic and inorganic chemicals, is considered to be a major source of environmental pollution. A random screen of 20 organic man-made chemicals present in liquid effluents revealed that half appeared able to interact with the estradiol receptor. This was demonstrated by their ability to inhibit binding of 17 beta-estradiol to the fish estrogen receptor. Further studies, using mammalian estrogen screens in vitro, revealed that the two phthalate esters butylbenzyl phthalate (BBP) and di-n-butylphthalate (DBP) and a food antioxidant, butylated hydroxyanisole (BHA) were estrogenic; however, they were all less estrogenic than the environmental estrogen octylphenol. Phthalate esters, used in the production of various plastics (including PVC), are among the most common industrial chemicals. Their ubiquity in the environment and tendency to bioconcentrate in animal fat are well known. Neither BBP nor DBP were able to act as antagonists, indicating that, in the presence of endogenous estrogens, their overall effect would be cumulative. Recently, it has been suggested that environmental estrogens may be etiological agents in several human diseases, including disorders of the male reproductive tract and breast and testicular cancers. The current finding that some phthalate compounds and some food additives are weakly estrogenic in vitro, needs to be supported by further studies on their effects in vivo before any conclusions can be made regarding their possible role in the development of these conditions.

Until recently, steroid hormones were believed to act only on cells containing intracellular receptors. However, recent evidence suggests that steroids have specific and rapid effects at the cellular membrane. Using whole-cell patch-clamp techniques, 17 beta-estradiol was found to reduce Ba2+ entry reversibly via Ca2+ channels in acutely dissociated and cultured neostriatal neurons. The effects were sex-specific, i.e., the reduction of Ba2+ currents was greater in neurons taken from female rats. 17 beta-Estradiol primarily targeted L-type currents, and their inhibition was detected reliably within seconds of administration. The maximum reduction by 17 beta-estradiol occurred at picomolar concentrations. 17 beta-Estradiol conjugated to bovine serum albumin also reduced Ba2+ currents, suggesting that the effect occurs at the membrane surface. Dialysis with GTP gamma S prevented reversal of the modulation, suggesting that 17 beta-estradiol acts via G-protein activation. 17 alpha-Estradiol also reduced Ba2+ currents but was significantly less effective than 17 beta-estradiol. Estriol and 4-hydroxyestradiol were found to reduce Ba2+ currents with similar efficacy to 17 beta-estradiol, whereas estrone and 2-methoxyestriol were less effective. Tamoxifen also reduced Ba2+ currents but did not occlude the effect of 17 beta-estradiol. These results suggest that at physiological concentrations, 17 beta-estradiol can have immediate actions on neostriatal neurons via nongenomic signaling pathways.

BACKGROUND

Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins.

RESULTS

Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G.

CONCLUSIONS

S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.

Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the alpha mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by alpha cells and is transcribed only in alpha cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First, wild-type Ste3 product is required for response to the pheromone: mutants with any one of five different ste3 mutations are unresponsive to a factor. Second, a hybrid Ste3-beta-galactosidase protein encoded by a STE3-lacZ gene fusion fractionates to the particulate fraction of yeast cell extracts, suggesting that Ste3 is a membrane protein. Finally, the DNA sequence of STE3, which we report here, encodes a protein of 470 amino acid residues that contains seven distinct hydrophobic segments of sufficient length to span a lipid bilayer.

Human cancer risks are inversely correlated with (a) blood retinol and (b) dietary beta-carotene. Although retinol in the blood might well be truly protective, this would be of little immediate value without discovery of the important external determinants of blood retinol which (in developed countries) do not include dietary retinol or beta-carotene. If dietary beta-carotene is truly protective--which could be tested by controlled trials--there are a number of theoretical mechanisms whereby it might act, some of which do not directly involve its 'provitamin A' activity.

The locations of HIV-1 RT nucleoside and non-nucleoside inhibitor-binding sites and inhibitor-resistance mutations are analyzed in the context of the three-dimensional structure of the enzyme and implications for mechanisms of drug inhibition and resistance are discussed. In order to help identify residues that may play a role in inhibitor binding, solvent accessibilities of amino acids that comprise the inhibitor-binding sites in the structure of HIV-1 RT complexed with a dsDNA template-primer are analyzed. While some mutations that cause resistance to nucleoside analogs, such as AZT, ddI, and ddC, are located near enough to the dNTP-binding site to directly interfere with binding of nucleoside analogs, many are located away from the dNTP-binding site and more likely confer resistance by other mechanisms. Many of the latter mutations are located on the surface of the DNA-binding cleft and may lead to altered template-primer positioning or conformation, causing a distortion of the geometry of the polymerase active site and consequent discrimination between normal and altered dNTP substrates. Other nucleoside analog-resistance mutations located on the periphery of the dNTP-binding site may exert their effects via altered interactions with dNTP-binding site residues. The structure of the hydrophobic region in HIV-1 RT that binds non-nucleoside inhibitors, for example, nevirapine and TIBO, has been analyzed in the absence of bound ligand. The pocket that is present when non-nucleoside inhibitors are bound is not observed in the inhibitor-free structure of HIV-1 RT with dsDNA. In particular it is filled by Tyr181 and Tyr188, suggesting that the pocket is formed primarily by rotation of these large aromatic side-chains. Existing biochemical data, taken together with the three-dimensional structure of HIV-1 RT, makes it possible to propose potential mechanisms of inhibition by non-nucleoside inhibitors. One such mechanism is local distortion of HIV-1 RT structural elements thought to participate in catalysis: the beta 9-beta 10 hairpin (which contains polymerase active site residues) and the beta 12-beta 13 hairpin ("primer grip"). An alternative possibility is restricted mobility of the p66 thumb subdomain, which is supported by the observation that structural elements of the non-nucleoside inhibitor-binding pocket may act as a "hinge" for the thumb.(ABSTRACT TRUNCATED AT 400 WORDS)

The release of insulin from pancreatic islets requires negative regulation to ensure low levels of insulin release under resting conditions, as well as positive regulation to facilitate robust responsiveness to conditions of elevated fuel or glucose. The first phase of release involves the plasma-membrane fusion of a small pool of granules, termed the readily releasable pool; these granules are already at the membrane under basal conditions, and discharge their cargo in response to nutrient and also non-nutrient secretagogues. By contrast, second-phase secretion is evoked exclusively by nutrients, and involves the mobilization of intracellular granules to t-SNARE sites at the plasma membrane to enable the distal docking and fusion steps of insulin exocytosis. Nearly 40 years ago, the actin cytoskeleton was first recognized as a key mediator of biphasic insulin release, and was originally presumed to act as a barrier to block granule docking at the cell periphery. More recently, however, the discovery of cycling GTPases that are involved in F-actin reorganization in the islet beta-cell, combined with the availability of reagents that are more specific and tools with which to study the mechanisms that underlie granule movement, have contributed greatly to our understanding of the role of the cytoskeleton in regulating biphasic insulin secretion. Herein, we provide historical perspective and review recent progress that has been made towards integrating cytoskeletal reorganization and cycling of small Rho-, Rab- and Ras-family GTPases into our current models of stimulus-secretion coupling and second-phase insulin release.

p27kip1 (p27) is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. p27 expression is regulated by cell contact inhibition and by specific growth factors, such as transforming growth factor (TGF)-beta. Since the cloning of the p27 gene in 1994, a host of other functions have been associated with this cell cycle protein. In addition to its role as a CDKI, p27 is a putative tumor suppressor gene, regulator of drug resistance in solid tumors, and promoter of apoptosis; acts as a safeguard against inflammatory injury; and has a role in cell differentiation. The level of p27 protein expression decreases during tumor development and progression in some epithelial, lymphoid, and endocrine tissues. This decrease occurs mainly at the post-translational level with protein degradation by the ubiquitin-proteasome pathway. A large number of studies have characterized p27 as an independent prognostic factor in various human cancers, including breast, colon, and prostate adenocarcinomas. Here we review the role of p27 in the regulation of the cell cycle and other cell functions and as a diagnostic and prognostic marker in human neoplasms. We also review studies indicating the increasingly important roles of p27, other CDKIs, and cyclins in endocrine cell hyperplasia and tumor development.

BACKGROUND

Vascular smooth muscle cell proliferation plays an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipocyte-specific plasma protein, accumulated in the human injured artery and suppressed endothelial inflammatory response as well as macrophage-to-foam cell transformation. The present study investigated the effects of adiponectin on proliferation and migration of human aortic smooth muscle cells (HASMCs). Methods and Results- HASMC proliferation was estimated by [(3)H] thymidine uptake and cell number. Cell migration assay was performed using a Boyden chamber. Physiological concentrations of adiponectin significantly suppressed both proliferation and migration of HASMCs stimulated with platelet-derived growth factor (PDGF)-BB. Adiponectin specifically bound to (125)I-PDGF-BB and significantly inhibited the association of (125)I-PDGF-BB with HASMCs, but no effects were observed on the binding of (125)I-PDGF-AA or (125)I-heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) to HASMCs. Adiponectin strongly and dose-dependently suppressed PDGF-BB-induced p42/44 extracellular signal-related kinase (ERK) phosphorylation and PDGF beta-receptor autophosphorylation analyzed by immunoblot. Adiponectin also reduced PDGF-AA-stimulated or HB-EGF-stimulated ERK phosphorylation in a dose-dependent manner without affecting autophosphorylation of PDGF alpha-receptor or EGF receptor.

CONCLUSIONS

The adipocyte-derived plasma protein adiponectin strongly suppressed HASMC proliferation and migration through direct binding with PDGF-BB and generally inhibited growth factor-stimulated ERK signal in HASMCs, suggesting that adiponectin acts as a modulator for vascular remodeling.

The prion hypothesis proposes that proteins can act as infectious agents. Originally formulated to explain transmissible spongiform encephalopathies (TSEs), the prion hypothesis has been extended with the finding that several non-Mendelian traits in fungi are due to heritable changes in protein conformation, which may in some cases be beneficial. Although much remains to be learned about the specific role of cellular cofactors, mechanistic parallels between the mammalian and yeast prion phenomena point to universal features of conformation-based infection and inheritance involving propagation of ordered beta-sheet-rich protein aggregates commonly referred to as amyloid. Here we focus on two such features and discuss recent efforts to explain them in terms of the physical properties of amyloid-like aggregates. The first is prion strains, wherein chemically identical infectious particles cause distinct phenotypes. The second is barriers that often prohibit prion transmission between different species. There is increasing evidence suggesting that both of these can be manifestations of the same phenomenon: the ability of a protein to misfold into multiple self-propagating conformations. Even single mutations can change the spectrum of favored misfolded conformations. In turn, changes in amyloid conformation can shift the specificity of propagation and alter strain phenotypes. This model helps explain many common and otherwise puzzling features of prion inheritance as well as aspects of noninfectious diseases involving toxic misfolded proteins.

Adoptive T-cell therapy (ACT) is a potent and flexible cancer treatment modality that can induce complete, durable regression of certain human malignancies. Long-term follow-up of patients receiving tumor-infiltrating lymphocytes (TILs) for metastatic melanoma reveals a substantial subset that experienced complete, lasting tumor regression - and may be cured. Increasing evidence points to mutated gene products as the primary immunological targets of TILs from melanomas. Recent technological advances permit rapid identification of the neoepitopes resulting from these somatic gene mutations and of T cells with reactivity against these targets. Isolation and adoptive transfer of these T cells may improve TIL therapy for melanoma and permit its broader application to non-melanoma tumors. Extension of ACT to other malignancies may also be possible through antigen receptor gene engineering. Tumor regression has been observed following transfer of T cells engineered to express chimeric antigen receptors against CD19 in B-cell malignancies or a T-cell receptor against NY-ESO-1 in synovial cell sarcoma and melanoma. Herein, we review recent clinical trials of TILs and antigen receptor gene therapy for advanced cancers. We discuss lessons from this experience and consider how they might be applied to realize the full curative potential of ACT.

Dorsal dermis and epaxial muscle have been shown to arise from the central dermomyotome in the chick. En1 is a homeobox transcription factor gene expressed in the central dermomyotome. We show by genetic fate mapping in the mouse that En1-expressing cells of the central dermomyotome give rise to dorsal dermis and epaxial muscle and, unexpectedly, to interscapular brown fat. Thus, the En1-expressing central dermomyotome normally gives rise to three distinct fates in mice. Wnt signals are important in early stages of dermomyotome development, but the signal that acts to specify the dermal fate has not been identified. Using a reporter transgene for Wnt signal transduction, we show that the En1-expressing cells directly underneath the surface ectoderm transduce Wnt signals. When the essential Wnt transducer beta-catenin is mutated in En1 cells, it results in the loss of Dermo1-expressing dorsal dermal progenitors and dermis. Conversely, when beta-catenin was activated in En1 cells, it induces Dermo1 expression in all cells of the En1 domain and disrupts muscle gene expression. Our results indicate that the mouse central dermomyotome gives rise to dermis, muscle, and brown fat, and that Wnt signalling normally instructs cells to select the dorsal dermal fate.

The R-Spondin (RSpo) family of secreted proteins act as potent activators of the Wnt/beta-catenin signaling pathway. We have previously shown that RSpo proteins can induce proliferative effects on the gastrointestinal epithelium in mice. Here we provide a mechanism whereby RSpo1 regulates cellular responsiveness to Wnt ligands by modulating the cell-surface levels of the coreceptor LRP6. We show that RSpo1 activity critically depends on the presence of canonical Wnt ligands and LRP6. Although RSpo1 does not directly activate LRP6, it interferes with DKK1/Kremen-mediated internalization of LRP6 through an interaction with Kremen, resulting in increased LRP6 levels on the cell surface. Our results support a model in which RSpo1 relieves the inhibition DKK1 imposes on the Wnt pathway.

A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble beta-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble beta-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 microg/ml) and anti-human IL-6 (10 microg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 microM) and its immunomodulatory analog IMiD1-CC4047 (1 microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.

BACKGROUND

Visfatin (also known as pre-B cell colony-enhancing factor or PBEF) is a cytokine that is highly expressed in visceral fat and whose blood levels correlate with obesity. Originally isolated as a secreted factor that promotes the growth of B cell precursors and recently found to act as an insulin analog on the insulin receptor, its pathophysiological role in humans remains largely unknown.

OBJECTIVE

In this study we investigated whether plasma visfatin level is altered in patients with type 2 diabetes mellitus (T2DM).

METHODS

Plasma visfatin as well as adiponectin and resistin concentrations were measured through ELISA in type 2 diabetic and nondiabetic subjects.

RESULTS

A total of 61 patients with T2DM and 59 sex- and age-matched nondiabetic subjects were studied. Plasma visfatin was found to be elevated in patients with T2DM (31.9 +/- 31.7 vs. 15.8 +/- 16.7 ng/ml, P = 0.002). In contrast, adiponectin was decreased (4.3 +/- 2.5 vs. 30.8 +/- 10.3 microg/ml, P < 0.001), whereas plasma resistin level did not differ between the groups. Increasing concentrations of visfatin were independently and significantly associated with T2DM. Multiple logistic regression analysis revealed visfatin as an independent association factor for T2DM, even after full adjustment of known biomarkers. The association between adiponectin and T2DM was no longer significant after adjustments for body mass index or waist to hip ratio. In a multiple linear regression analysis, only waist to hip ratio was independently associated with plasma visfatin level.

CONCLUSIONS

Our results indicate that visfatin may play a role in the pathogenesis of T2DM.

Insulin-like growth f<em>act</em>or (IGF) binding protein-3 (IGFBP-3) is known to block IGF <em>act</em>ion and inhibit cell growth. IGFBP-3 is thought to <em>act</em> by sequestering free IGFs or, possibly, <em>act</em> via a novel IGF-independent mechanism. Supporting its role as a primary growth inhibitor, IGFBP-3 production has been shown to be increased by cell growth-inhibitory agents, such as transforming growth f<em>act</em>or-<em>beta</em> (TGF-<em>beta</em>), and the tumor suppressor gene p53. In this paper, we demonstrate, for the first time, a novel function of IGFBP-3 as an apoptosis-inducing agent and show that this <em>act</em>ion is mediated through an IGF.IGF receptor-independent pathway. In the p53 negative prostate cancer cell line, PC-3, the addition of recombinant IGFBP-3 resulted in a dose-dependent induction of apoptosis. 125I-IGFBP-3 bound with high affinity to specific proteins in PC-3 cell lysates and plasma membrane preparations. These membrane-associated molecules may serve as receptors that mediate the direct effect of IGFBP-3 on apoptosis. In addition, in an IGF receptor-negative mouse fibroblast cell line, treatment with recombinant IGFBP-3 as well as transfection of the IGFBP-3 gene induced apoptosis, suggesting that neither IGFs nor IGF receptors are required for this <em>act</em>ion. Furthermore, treatment with TGF-<em>beta</em>1, a known apoptosis-inducing agent, resulted in the induction of IGFBP-3 expression 6-12 h before the onset of apoptosis. This effect of TGF-<em>beta</em>1 was prevented by co-treatment with IGFBP-3-neutralizing antibodies or IGFBP-3-specific antisense thiolated oligonucleotides. These findings suggest that IGFBP-3 induces apoptosis through a novel pathway independent of either p53 or the IGF.IGF receptor-mediated cell survival pathway and that IGFBP-3 mediates TGF-<em>beta</em>1 induced apoptosis in PC-3 cells.

Branching morphogenesis in the lung serves as a model for the complex patterning that is reiterated in multiple organs throughout development. Beta-catenin and Wnt signaling mediate critical functions in cell fate specification and differentiation, but specific functions during branching morphogenesis have remained unclear. Here, we show that Wnt/beta-catenin signaling regulates proximal-distal differentiation of airway epithelium. Inhibition of Wnt/beta-catenin signaling, either by expression of Dkk1 or by tissue-specific deletion of beta-catenin, results in disruption of distal airway development and expansion of proximal airways. Wnt/beta-catenin functions upstream of BMP4, FGF signaling, and N-myc. Moreover, we show that beta-catenin and LEF/TCF activate the promoters of BMP4 and N-myc. Thus, Wnt/beta-catenin signaling is a critical upstream regulator of proximal-distal patterning in the lung, in part, through regulation of N-myc, BMP4, and FGF signaling.

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits. In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively. By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways. Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma. This effect of beta gamma requires costimulation by alpha s-GTP. This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active. We now confirm this prediction. Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis. Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation. These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.