Defective WNT signaling associates with bone marrow fibrosis-a cross-sectional cohort study in a family with WNT1 osteoporosis.

This study explores bone marrow function in patients with defective WNT1 signaling. Bone marrow samples showed increased reticulin and altered granulopoiesis while overall hematopoiesis was normal. Findings did not associate with severity of osteoporosis. These observations provide new insight into the role of WNT signaling in bone marrow homeostasis. INTRODUCTION: WNT signaling regulates bone homeostasis and survival and self-renewal of hematopoietic stem cells. Aberrant activation may lead to osteoporosis and bone marrow pathology. We aimed to explore bone marrow findings in a large family with early-onset osteoporosis due to a heterozygous WNT1 mutation. METHODS: We analyzed peripheral blood samples, and bone marrow aspirates and biopsies from 10 subjects with WNT1 mutation p.C218G. One subject was previously diagnosed with idiopathic myelofibrosis and others had no previously diagnosed hematologic disorders. The findings were correlated with the skeletal phenotype, as evaluated by number of peripheral and spinal fractures and bone mineral density. RESULTS: Peripheral blood samples showed no abnormalities in cell counts, morphology or distributions but mild increase in platelet count. Bone marrow aspirates (from 8/10 subjects) showed mild decrease in bone marrow iron storages in 6 and variation in cell distributions in 5 subjects. Bone marrow biopsies (from 6/10 subjects) showed increased bone marrow reticulin (grade MF-2 in the myelofibrosis subject and grade MF-1 in 4 others), and an increase in overall, and a shift towards early-phase, granulopoiesis. The bone marrow findings did not associate with the severity of skeletal phenotype. CONCLUSIONS: Defective WNT signaling associates with a mild increase in bone marrow reticulin and may predispose to myelofibrosis, while overall hematopoiesis and peripheral blood values are unaltered in individuals with a WNT1 mutation. In this family with WNT1 osteoporosis, bone marrow findings were not related to the severity of osteoporosis.

Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion.

Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.

New approaches to investigating heterogeneity in complex traits.

Great advances in the field of genetics have been made in the last few years. However, resolving the complexity that underlies the susceptibility to many polygenic human diseases remains a major challenge to researchers. The fast increase in availability of genetic data and the better understanding of the clinical and pathological heterogeneity of many autoimmune diseases such as multiple sclerosis, but also Parkinson's disease, Alzheimer's disease, and many more, have changed our views on their pathogenesis and diagnosis, and begins to influence clinical management. At the same time, more powerful methods that allow the analysis of large numbers of genes and proteins simultaneously open opportunities to examine their complex interactions. Using multiple sclerosis as a prototype, we review here how new methodologies such as gene expression profiling can be exploited to gain insight into complex trait diseases.

JAB/SOCS1/SSI-1 is an interleukin-2-induced inhibitor of IL-2 signaling.

JAB/suppressor of cytokine signaling 1 (SOCS1) STAT-induced STAT inhibitor-1 (SSI-1) (JAB/SOCS1/SSI-1) is an SH2-domain-containing protein that is induced by and negatively regulates signaling by a number of cytokines including interleukin-4 (IL-4), IL-6, interferon (IFN)-gamma, prolactin, growth hormone, and erythropoietin. The role of JAB/SOCS1/SSI-1 in IL-2 signaling has been analyzed. JAB/SOCS1/SSI-1 is strongly induced by IL-2 in peripheral blood T cells, and JAB/SOCS1/SSI-1 overexpression strongly inhibits IL-2-induced signal transducer and activator of transcription-5 (Stat5) phosphorylation and transcriptional activity. In cotransfection experiments, JAB/SOCS1/SSI-1 associates with both Jak1 and Jak3; however, JAB/SOCS1/SSI-1 had a greater effect on Jak1 tyrosine phosphorylation and kinase activity. JAB/SOCS1/SSI-1 also interacts with IL-2Rbeta, and this interaction requires the A region (residues 313-382) of IL-2Rbeta. However, this interaction was not essential for the inhibitory action of JAB. Thus, JAB/SOCS1/SSI-1 is an IL-2-induced inhibitor of IL-2 signaling that functions by inhibiting Jak kinase activity. This suggests an important role for JAB/SOCS1/SSI-1 in regulating T-cell responses.

Regulation of Jak2 tyrosine kinase by protein kinase C during macrophage differentiation of IL-3-dependent myeloid progenitor cells.

Differentiation of macrophages from myeloid progenitor cells depends on a discrete balance between cell growth, survival, and differentiation signals. Interleukin-3 (IL-3) supports the growth and survival of myeloid progenitor cells through the activation of Jak2 tyrosine kinase, and macrophage differentiation has been shown to be regulated by protein kinase C (PKC). During terminal differentiation of macrophages, the cells lose their mitogenic response to IL-3 and undergo growth arrest, but the underlying signaling mechanisms have remained elusive. Here we show that in IL-3-dependent 32D myeloid progenitor cells, the differentiation-inducing PKC isoforms PKC-alpha and PKC-delta specifically caused rapid inhibition of IL-3-induced tyrosine phosphorylation. The target for this inhibition was Jak2, and the activation of PKC by 12-O-tetradecanoyl-phorbol-13-acetate treatment also abrogated IL-3-induced tyrosine phosphorylation of Jak2 in Ba/F3 cells. The mechanism of this regulation was investigated in 32D and COS7 cells, and the inhibition of Jak2 required catalytic activity of PKC-delta and involved the phosphorylation of Jak2 on serine and threonine residues by the associated PKC-delta. Furthermore, PKC-delta inhibited the in vitro catalytic activity of Jak2, indicating that Jak2 was a direct target for PKC-delta. In 32D cells, the inhibition of Jak2 either by PKC-delta, tyrosine kinase inhibitor AG490, or IL-3 deprivation caused a similar growth arrest. Reversal of PKC-delta-mediated inhibition by the overexpression of Jak2 promoted apoptosis in differentiating 32D cells. These results demonstrate a PKC-mediated negative regulatory mechanism of cytokine signaling and Jak2, and they suggest that it serves to integrate growth-promoting and differentiation signals during macrophage differentiation. (Blood. 2000;95:1626-1632)

Interleukin-4-induced transcriptional activation by stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6.

Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3'-kinase pathway. Phospho-amino acid analysis and tryptic phosphopeptide maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway. (Blood. 2000;95:494-502)

Inhibitors keep cytokines in check.

Some members of the CIS/SOCS/JAB/SSI family have been demonstrated to be cytokine-inducible inhibitors of cytokine signaling. Steps have now been made towards clarifying the biological function of two of these proteins, revealing that these inhibitors are essential for the correct maintenance of cytokine signaling.

c-Myc and E1A induced cellular sensitivity to activated NK cells involves cytotoxic granules as death effectors.

The contact of natural killer (NK) cells with foreign cells and with certain virus-infected or tumor cells triggers the cytolytic machinery of NK cells. This triggering leads to exocytosis of the cytotoxic NK cell granules. The oncoproteins c-Myc and E1A render cells vulnerable to NK cell mediated cytolysis yet the mechanisms of sensitization are not well understood. In a model where foreign cells (rat fibroblasts) were cocultured with human IL-2 activated NK cells, we observed that NK cells were capable of efficiently killing their targets only if the cells overexpressed the oncogene c-Myc or E1A. Both the parental and the oncogene expressing fibroblasts similarly triggered phosphoinositide hydrolysis in the bound NK cells, demonstrating that NK cells were cytolytically activated in contact with both resistant parental and oncogene expressing sensitive target fibroblasts. The cell death was independent of wild-type p53 and was not inhibited by an anti-apoptotic protein EIB19K. These results provided evidence that c-Myc and E1A activated the NK cell induced cytolysis at a post-triggering stage of NK cell-target cell interaction. In consistence, the c-Myc and E1A overexpressing fibroblasts were more sensitive to the cytolytic effects of isolated NK cell-derived granules than parental cells. The data indicate that oncogenes activate the cytotoxicity of NK cell granules. This mechanism can have a role in directing the cytolytic action of NK cells towards the virus-infected and cancer cells.

Lymphocyte adhesion molecule ligands and extracellular matrix proteins in gliomas and normal brain: expression of VCAM-1 in gliomas.

To identify antigenic differences between gliomas and normal brain, we have immunohistochemically studied the expression of lymphocyte adhesion molecules (ICAM-1, ICAM-2, ICAM-3, VCAM-1, E-selectin and CD58), epidermal growth factor receptor (EGFR) and extracellular matrix proteins (collagen IV, fibronectin, laminin, merosin, tenascin and vitronectin) in these tissues. Gliomas expressed high levels of ICAM-1, CD58 (LFA-3), EGFR, tenascin and vitronectin, whereas only very low levels were detected in normal brain. VCAM-1 expression was detected in 15 out of 25 gliomas but not in normal brain. The presence of VCAM-1 in gliomas was verified by immunoblotting and RNase protection assay, and in glioma cell lines by Northern blotting. Expression of VCAM-1 in gliomas may partially explain lymphocytic infiltration, and anti-VCAM-1 antibodies may be of potential in antibody mixtures for targeted therapy of gliomas.

ICAM-2 redistributed by ezrin as a target for killer cells.

Very little is known about the receptors and target molecules involved in natural killer (NK) cell activity. Here we present a model system in which interleukin-2-activated killing by NK cells depends on the intercellular adhesion molecule ICAM-2 and is regulated by the distribution of ICAM-2. The level of ICAM-2 expression in NK-sensitive and resistant cells is similar, but in sensitive cells ICAM-2 is concentrated into bud-like cellular projections known as uropods, whereas in resistant cells it is evenly distributed. The cytoskeletal-membrane linker protein ezrin is also localized in uropods. Transfection of human ezrin into NK-resistant cells induces uropods formation, redistribution of ICAM-2 and ezrin, and sensitizes target cells to interleukin-2-activated killing. These results reveal a new mechanism of target-cell recognition: cytotoxic cells recognize adhesion molecules that are already present on normal cells, but in diseased cells are concentrated into a biologically active cell-surface region by cytoskeletal reorganization. The results also highlight the importance of cytoskeletal interactions in the regulation of ICAM-2-mediated adhesive phenomena.

Expression and characterization of a B cell growth promoting polypeptide derived from the 12 kDa B cell growth factor gene (BCGF 1).

The expression and partial purification of recombinant 12 kDa B cell growth factor are reported. The polypeptide was derived from the genomic sequence of the gene (BCGF 1) which is here shown to be a single copy gene that localizes to human chromosome 16. When expressed as a glutathione S-transferase fusion protein in E. coli, the protein appears as a 38 kDa polypeptide in Western blot analysis using a peptide antibody. The purified fusion protein stimulates the proliferation of activated human B cells in a dose-dependent manner, and the active site resides within the 104 carboxy-terminal amino acids. The availability of biologically active recombinant 12 kDa B cell growth factor will enable its evaluation in B cell growth regulation, and provides a new means of in vitro culturing of human B lymphocytes.

Requirement of CD4+ lymphocytes in IL-2-stimulated NK cell proliferation.

Growth requirements of human natural killer cells in IL-2-supplemented cultures were studied. NK cell proliferation was monitored by the MAC (morphology antibody chromosomes) technique and subset specific cell cycle analysis, which both enable direct determination of cell growth in specific lymphocyte subsets among heterogeneous lymphocyte populations. Our results show that even in the presence of saturating concentrations of IL-2, the proliferative capacity of purified CD16+ cells is quite low, but can be stimulated in a dose dependent manner by CD4+ cells. CD4+ cells could partially be replaced by IL-4 but not by various other commercially available cytokines. These results provide further evidence of the requirement of accessory stimuli in NK cell proliferation, and support the interpretation that NK cells have a direct regulatory role in specific T cell responses.

Philadelphia chromosome as the sole abnormality and p210 bcr-abl chimeric protein expression in an Epstein-Barr virus-transformed B cell line from a patient with chronic myeloid leukemia.

A 'B' cell line, originating from a patient with chronic myeloid leukemia and containing the Philadelphia chromosome, was established after Epstein-Barr virus transformation. The Philadelphia chromosome was the sole chromosomal abnormality in this line, designated as PhB1 cell line. In DNA hybridization studies we detected rearrangements in the bcr gene and in the immunoglobulin heavy chain joining region. The phenotypes of the cells were typical of mature B cells expressing antigens CD19, CD20, CD22, CD23, CD39, HLADR, IgM, and kappa. The expression of the 210 bcr-abl chimeric protein was detected by means of an immunoprecipitate assay.

Effect of a low-molecular-weight B cell growth factor on the proliferation of normal and neoplastic lymphocytes in lymphomas.

The usefulness of low-molecular-weight B-cell growth factor (LMW-BCGF) for routine cytogenetic study of B cell non-Hodgkin lymphomas (NHL) was evaluated. Eighteen B cell lymphoma specimens were cultured for 4 days in the presence or absence of LMW-BCGF. After culture, conventional cytogenetic analysis was carried out, and the Morphology Antibody Chromosomes (MAC) method was used to study the frequencies of mitotic cells in different lymphocyte subsets. Our results showed that in 6 of the 14 lymphomas with a chromosomally abnormal clone LMW-BCGF produced an increased frequency of abnormal mitosis. In eight cases, LMW-BCGF did not increase, or even decrease, the frequency of karyotypically abnormal mitoses. In these eight cases, the total number of mitosis was often increased, but the mitotic cells were shown to be T cells. LMW-BCGF seems to have a clearly favorable effect on the cytogenetics of some B cell NHL, but apparently it is not a radical improvement over the routine techniques now being used.

MAC technique (morphology antibody chromosomes) in phenotypic identification of proliferating NK and T cells in interleukin-2-stimulated lymphocyte cultures.

Human peripheral blood lymphocytes were activated with recombinant interleukin 2 (rIL-2) and cultured in fetal calf serum (FCS)- or human-AB-serum-supplemented media. Proliferative cells were identified by the MAC technique (morphology antibody chromosomes) which enables the immunoenzymatic identification of both mitotic and non-proliferating cells in unfractionated lymphocyte populations. The results indicate that the phenotype of more than 90% of proliferative lymphocytes can be characterized by using antibodies against T cells and NK cells. Substantial mitotic activity of CD4-positive (CD4+) T cells was observed only in FCS-supplemented cultures, whereas in serum-free or human-AB-serum-supplemented cultures mostly CD8+ T cells and NK cells proliferated. The proportion of NK cells among all mitotic cells varied between 14 and 32%. Interestingly, in unfractionated cultures approximately 13% NK cells entered mitoses in the presence of rIL-2, suggesting that the poor proliferative capacity of purified NK cells demonstrated previously may be due to the lack of accessory stimulatory signals. The proliferation of B cells was minimal in all experiments. The MAC technique is a useful addition to the techniques by which lymphocyte growth regulation is monitored.

Mitotic cells in different lymphocyte subsets in unfractionated cultures stimulated by phytohaemagglutinin or pokeweed mitogen.

Unfractionated peripheral blood lymphocytes were cultured in the presence of either phytohaemagglutinin (PHA) or pokeweed mitogen (PWM) for 3 and 6 days. The subpopulations of mitotic lymphocytes were determined by an immunoperoxidase technique. Our results demonstrate that the proportion of mitotic B cells is approximately equal in PHA- and PWM-stimulated cultures. The frequency of mitotic CD22+ B cells varied from 3% to 5% and that of cells positive for the monoclonal antibodies anti-lambda and anti-kappa from 5% to 7%. We also showed that PHA stimulates mitoses in CD8-positive suppressor cells more effectively than does PWM. The mean proportion of CD8+ cells in mitosis was 29% when cultured with PHA for 3 days and 17%, in the presence of PWM. Culturing for 6 days with PHA increased the proportion of mitotic CD8+ cells, but PWM stimulation did not affect the frequency of mitoses in this lymphocyte subgroup.

Relative proportions of mitotic T and B cells in PHA-stimulated lymphocyte cultures.

Peripheral blood lymphocytes from six healthy adults were stimulated with phytohemagglutinin (PHA) and cultured for 3 days. The relative proportions of mitotic lymphocyte subsets were determined by a method that allows both cytogenetic and immunologic characterization of a mitotic cell. The results directly indicated that not only T cells but also B cells undergo mitosis in PHA-stimulated lymphocyte cultures. The frequency of the mitotic B cells varied between 6% and 20%.