VLA-5-mediated Adhesion to Fibronectin Accelerates Hemin-stimulated Erythroid Differentiation of K562 Cells through Induction of VLA-4 Expression

Fibronectin plays important roles in erythropoiesis through the fibronectin receptors VLA-4 and VLA-5. However, the substantial role of these fibronectin receptors and their functional assignment in erythroid differentiation are not yet fully understood. Here, we investigated the effects of cell adhesion to fibronectin on erythroid differentiation using K562 human erythroid progenitor cells. Erythroid differentiation could be induced in K562 cells in suspension by stimulating with hemin. This hemin-stimulated erythroid differentiation was highly accelerated when cells were induced to adhere to fibronectin by treatment with TNIIIA2, a peptide derived from tenascin-C, which has recently been found to induce β1-integrin activation. Another integrin activator, Mn²⁺, also accelerated hemin-stimulated erythroid differentiation. Adhesive interaction with fibronectin via VLA-4 as well as VLA-5 was responsible for acceleration of the hemin-stimulated erythroid differentiation in response to TNIIIA2, although K562 cells should have been lacking in VLA-4. Adhesion to fibronectin forced by TNIIIA2 causally induced VLA-4 expression in K562 cells, and this was blocked by the RGD peptide, an antagonist for VLA-5. The resulting adhesive interaction with fibronectin via VLA-4 strongly enhanced the hemin-stimulated activation of p38 mitogen-activated protein kinase, which was shown to serve as a signaling molecule crucial for erythroid differentiation. Suppression of VLA-4 expression by RNA interference abrogated acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2. Thus, VLA-4 and VLA-5 may contribute to erythropoiesis at different stages of erythroid differentiation.Hematopoietic stem and progenitor cells proliferate and differentiate in the bone marrow and fetal liver (1–6). Stromal cells of the bone marrow and fetal liver form a hematopoietic microenvironment called a “niche.” This microenvironment niche plays a crucial role in the regulation of the proliferation and differentiation of hematopoietic stem and progenitor cells. Besides humoral factors that include hematopoietic growth factors, adhesive interaction of hematopoietic stem and progenitor cells with stromal cells and/or the extracellular matrix (ECM)² in the hematopoietic microenvironment is indispensable for hematopoietic development (1–6). The ECM in the hematopoietic microenvironment is composed of various macromolecules, such as fibronectin (FN), collagens, laminins, and proteoglycans. Among them, FN is one of the most important parts of the microenvironment niche (7–11). Also, in erythropoiesis, the importance of the adhesion of erythroid progenitors to FN via the FN receptors VLA-4 and VLA-5 has been reported (11–16). However, the substantial role of these FN receptors and their functional assignment in erythroid differentiation are not yet fully understood.We previously found that FN, which provides scaffolding for the adhesion of various cell types, has an alternative functional site opposing cell adhesion (17). A 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat of the FN molecule, termed FNIII14, strongly suppresses cell adhesion to FN by inhibiting the activation of β1-integrins including VLA-4 and VLA-5 (18, 19). Conversely, we have recently found that tenascin (TN)-C, which is an anti-adhesive ECM protein (20, 21), has a functional site for stimulating cell adhesion to FN (22). A 22-mer peptide derived from the FNIII repeat A2 in the TN-C molecule, termed TNIIIA2, can induce the conformational change necessary for functional activation of FN receptors through binding with syndecan-4 (22, 23). The active sites of FNIII14 and TNIIIA2 appear to be cryptic in the molecular structures of FN and TN-C but are exposed by conformational change through interaction with other ECM molecules or by processing with matrix metalloproteinase-2 (22, 24). Thus, these functional sites found in FN and TN-C molecules, which act in opposition to their parental ECM proteins, may act as a negative feedback loop for preventing excessive cellular responses to these ECM proteins in biological processes with ECM rearrangement. In any case, FNIII14 and TNIIIA2 enable us to control, either negatively or positively, the adhesion of various cell types to FN.Various hematopoietic progenitor cell lines have been used in in vitro studies of hematopoietic differentiation. However, most hematopoietic progenitor cell lines are nonadherent, because their cell surface β1-integrins, including FN receptors, have impaired ligand-binding activity (25, 26). Therefore, in order to investigate the role of cell adhesion to FN in hematopoietic differentiation, their FN receptors must be activated. Since TNIIIA2 can induce activation of FN receptors in various hematopoietic progenitor cell lines (22), this peptide factor may be useful for investigating the substantial role of cell adhesion to FN in hematopoietic differentiation. Here, we investigate the effects of cell adhesion to FN on erythroid differentiation using TNIIIA2 and Mn²⁺ as the integrin activator and the human erythroid progenitor cell line K562, which only expresses VLA-5, as the FN receptor (27). As a result, we show that hemin-stimulated erythroid differentiation of K562 cells is strongly enhanced when K562 cells are forced to adhere to FN. Sustained adhesion to FN via VLA-5, which is induced by TNIIIA2 or Mn²⁺, causes induction of VLA-4 expression. The resulting adhesive interaction with FN via newly expressed VLA-4 then generates a conspicuous increase in the hemin-stimulated phosphorylation/activation of p38 MAP kinase, which is shown to serve as a signaling molecule crucial for erythroid differentiation of K562 cells.     

Palmitate Induces Insulin Resistance in H4IIEC3 Hepatocytes through Reactive Oxygen Species Produced by Mitochondria

Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH₂-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated β-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance.Insulin is the major hormone that inhibits gluconeogenesis in the liver. Visceral adiposity in obesity causes hepatic steatosis and insulin resistance. In an insulin-resistant state, impaired insulin action allows enhancement of glucose production in the liver, resulting in systemic hyperglycemia (1) and contributing to the development of type 2 diabetes. In addition, we have demonstrated experimentally that insulin resistance accelerated the pathology of steatohepatitis in genetically obese diabetic OLETF rats (2). In contrast, lipid-induced oxidative stress caused steatohepatitis and hepatic insulin resistance in mice (3). In fact, steatosis of the liver is an independent predictor of insulin resistance in patients with nonalcoholic fatty liver disease (4).It remains unclear whether hepatic steatosis causally contributes to insulin resistance or whether it is merely a resulting pathology. Excessive dietary free fatty acid (FFA)² flux into the liver via the portal vein may cause fatty liver disease and hepatic insulin resistance. Indeed, elevated plasma FFA concentrations correlate with obesity and decreased target tissue insulin sensitivity (5).Experimentally, lipid infusion or a high fat diet that increases circulating FFA levels promotes insulin resistance in the liver. Candidate events linking FFA to insulin resistance in vivo are the up-regulation of SREBP-1c (6), inflammation caused by activation of c-Jun amino-terminal kinase (JNK) (7) or IKKβ (8), endoplasmic reticulum (ER) stress (9), ceramide (10, 11), and TRB3 (12).However, which event is the direct and initial target of FFA in the liver is unclear. Insulin resistance induced by lipid infusion or a high fat diet is complex and may be accompanied by alterations not restricted to the liver, making it difficult to determine the contribution of FFAs to hepatic insulin resistance. For example, hyperinsulinemia and hyperglycemia secondary to the initial event also may contribute to the development of diet-induced insulin resistance in vivo (6).To address the early event(s) triggering the development of high fat diet- or obesity-induced insulin resistance, we investigated the molecular mechanism(s) underlying the direct action of FFA on hepatocytes to cause insulin resistance in vitro, using the rat hepatocyte cell line H4IIEC3. We found that mitochondria-derived reactive oxygen species (ROS) were a cause of palmitate-induced insulin resistance in hepatocytes.     

Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants

Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.     

Antitrypanosomal activity of some pregnane glycosides isolated from Caralluma species

Pregnane glycosides previously isolated from genus Caralluma (C. Penicillata, C. tuberculata and C. russelliana) were tested for their antitrypanosomal activity. Penicilloside E showed the highest antitrypanosomal activity (IC₅₀ 1.01 μg/ml) followed by caratuberside C (IC₅₀ 1.85 μg/ml), which exhibited the highest selectivity index (SI 12.04). It was noticed that acylation is required for the antitrypanosomal activity while glycosylation at C-20 has no significant effect on the activity.     

Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membranes

Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed.     

Creation of cross-linked electrospun isotypic-elastin fibers controlled cell-differentiation with new cross-linker

Effective application of elastin materials for vascular grafts in tissue engineering requires these materials to retain the elastic and biological properties of native elastin. To clarify the influence of soluble elastin isotypes on vascular smooth muscle cells (VSMCs), soluble elastin was prepared from insoluble elastin by hydrolysis with oxalic acid. Its fractions were separated and classified into three isotypes. Elastin retaining 2.25 mol% of cross-linked structures exhibited significant differentiation of VSMCs, which adhered to the elastin with contraction phenotypes similar to that of native elastin, causing proliferation to cease. This trend was more strongly demonstrated in cotton-like elastin fibers with a new cross-linker. The results suggest that elastin isotypes could be applied as new effective biomaterials for suppressing intimal hyperplasia in vascular grafts.