There is evidence that insulin-like growth factor-binding protein (IGFBP-2), a modulator of the actions of IGFs, also has IGF-independent effects in human tumor cell lines. These involve specific binding of IGFBP-2 to α5β1-integrin, followed by alterations in the phosphorylation status of downstream signaling molecules. Previously, IGFBP-2 has also been shown to be associated with cell proliferation, adhesion and migration. Here, we investigated direct effects of IGFBP-2 on apoptosis and alterations in the expression of related proteins. The breast cancer cell line Hs578T, which shows no IGFBP-2 production of its own and is independent of the IGF-I receptor, was treated with human recombinant IGFBP-2 in order to study the changes in gene expression induced by IGFBP-2. The methods employed for this purpose were oligonucleotide microarrays, real-time RT-PCR, western blotting, and immunoassays. Out of the 440 genes covered by the Oligo GEArray Human Cancer Microarray OHS-802, the expression of 77 genes was directly influenced by IGFBP-2. By the use of real-time quantitative RT-PCR, the gene expression of Nuclear Factor (NF)κB, p53, transforming growth factor β (TGF β-1), LAMB1 (Laminin, Beta 1), Bcl-2, and IIp45 was found to be significantly upregulated (by 1.2- to 3.05-fold; all P < 0.001). Accordingly, NFκB, p53, and TGF β-1 proteins, as measured by Western blotting and immunoassay, were upregulated > 1.5-fold. By using an ELISA-based and a flow cytometry-based apoptosis assay, IGFBP-2 was found to have a pro-apoptotic effect on Hs578T cells. Our results suggest that IGFBP-2-induced gene expressions are of functional significance for proliferation, cell adhesion, cell migration and apoptosis, and showed that IGFBP-2 can promote apoptosis in tumor cells independent of IGF.
Background— Bartonella species are the only known bacterial pathogens causing vasculoproliferative disorders in humans (bacillary angiomatosis [BA]). Cellular and bacterial pathogenetic mechanisms underlying the induction of BA are largely unknown. Methods and Results— Activation of hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in angiogenesis, was detected in Bartonella henselae –infected host cells in vitro by immunofluorescence, Western blotting, electrophoretic mobility shift, and reporter gene assays and by immunohistochemistry in BA tissue lesions in vivo. Gene microarray analysis revealed that a B henselae infection resulted in the activation of genes typical for the cellular response to hypoxia. HIF-1 was essential for B henselae –induced expression of vascular endothelial growth factor as shown by inhibition with the use of HIF-1–specific short-interfering RNA. Moreover, infection with B henselae resulted in increased oxygen consumption, cellular hypoxia, and decreased ATP levels in host cells. Infection with a pilus-negative variant of B henselae did not lead to cellular hypoxia or activation of HIF-1 or vascular endothelial growth factor secretion, suggesting a crucial role of this bacterial surface protein in the angiogenic reprogramming of the host cells. Conclusions— B henselae induces a proangiogenic host cell response via HIF-1. Our data provide for the first time evidence that HIF-1 may play a role in bacterial infections.
The IGF/IGF binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. Evidence from in vitro studies indicates that IGFBPs may have IGF-independent effects. We analyzed the biological activity of intact IGFBP-2 and defined carboxy-terminal IGFBP-2 fragments isolated from human hemofiltrate in two cell culture systems of the growth plate: rat growth plate chondrocytes in primary culture and the mesenchymal chondrogenic cell line RCJ3.1C5.18. The IGFBP-2 fragments IGFBP-2167–279, IGFBP-2167–289, and IGFBP-2104–289 exerted a strong (2- to 3-fold) mitogenic effect on growth plate chondrocytes, which was comparable with IGF-I in equimolar concentrations (7.8 nm) but was not mediated through the type 1 IGF receptor. In a dose-response experiment, the most effective concentration of IGFBP-2104–289 for the stimulation of cell proliferation was 10 nm. This biological activity of IGFBP-2 fragments was associated with cell membrane binding, demonstrated by Western blot analysis of fractionated cell lysates and immunohistochemistry. Whereas intact IGFBP-2 did not modulate chondrocyte proliferation, partially reduced (by dithiothreitol) full-length IGFBP-2 stimulated cell proliferation to a comparable extent (3.4-fold) as carboxy-terminal IGFBP-2 fragments. The mitogenic activity of these IGFBP-2 fragments and of partially reduced full-length IGFBP-2 was mediated through the use of the MAPK/ERK 1/2. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.
In vitro studies indicate that surface tension and surface viscosity of the tear film lipid layer (TFLL) are governed by interactions between meibomian lipids and proteins from the aqueous layer. The role of minor tear proteins with strong lipophilic properties or those correlated with pathological states is still unknown. The discovery of lung surfactant proteins (SPs) in tears and keratin in normal and abnormal meibomian gland excretions warrants investigation into their effects on the surface activity of meibomian lipid films.Commercial keratin and bovine lung SPs were used in vitro to assess the surface pressure of meibomian lipid films using a Langmuir trough.The pressure-area profiles of meibomian lipid films seeded with SPs (2.5 μL; 0.1 μg) demonstrated hybrid characteristics between meibomian lipid films alone and SPs alone but reached much higher maximum surface pressures (approximately 30 vs. 24 mN/m). Microscopically, the appearance of meibomian lipid films was not altered by SPs. Maximum surface pressure of meibomian films premixed with keratin was much higher than meibum alone. The pressure-area isocycles appeared more like those of meibomian lipids with a low concentration of protein and more like pure keratin films at a high concentration.The data strongly indicate that SPs and keratin likely interact with the TFLL. SPs are likely to act as strong surfactants and to reduce the surface tension of the lipid layer. Excess concentrations of keratin as identified in patients with meibomian gland dysfunction could disrupt the normal structure of the meibomian lipid film.
Minor histocompatibility antigens (miHags) are recognized by alloreactive cytotoxic donor T lymphocytes and trigger potent immune reactions such as graft-versus-host disease (GvHD) after major histocompatibility complex-matched transplantation. Our study focuses on tissue-specific T-cell responses to miHag-encoded peptides in GvHD target organs during the first 30 days in a murine transplant model.Complementarity determining region (CDR)3-size spectratyping was used to study T cell receptor (TCR) repertoires in recipient skin, liver, ileum, colon, spleen, and heart.GvHD occurred as early as day 14 and was proven by histology in skin, liver, ileum, and colon. The heart was histologically not affected by GvHD but showed endomyocardial "quilty lesions." Two distinct patterns of TCR diversities could be identified. In skin, a restricted V beta usage in combination with all J beta segments contrasted with a complete V beta repertoire in intestinal organs combined with a restricted J beta usage. Interestingly, TCR repertoire in the heart was almost identical with intestinal CDR3-size patterns. Persisting clones were found in skin from day 9 to 30. In intestine and heart, identical sequences were obtained from several organs on day 14 and 21, but no persistence of CDR3 sequences could be observed.These results suggest that in the skin a limited number of persisting T cell clones maintains GvHD, whereas in the intestine, temporary expansions of different clones may fuel the process of GvHD. Strategies that eliminate tissue-specific T cells on the basis of their activational status rather than their V beta expression but at the same time preserve a broad, overall TCR repertoire will help to increase the efficacy and safety of allogeneic stem cell transplantation.
