Inhibitory epidermal pentapeptide modulates proliferation and differentiation of transformed mouse epidermal cells in vitro.

A transformed mouse epidermal cell line ("308 cells") and nontransformed rat tongue squamous epithelial cells ("RT10 cells") were treated 3 times weekly for a period of two weeks with relatively large doses (150 micrograms/ml) of a synthetic inhibitory epidermal pentapeptide; pyroGlu-Glu-Asp-Ser-GlyOH. The peptide was recently isolated from mouse skin extracts and inhibits normal epidermal cells in vivo and in vitro at a restricted and low dose level. Repeated treatments with the large dose was followed by a 30-40% reduction in the number of 308 cells per well, starting as early as day 1. The number of RT10 cells was reduced about 20% only at termination of the experiment on day 14. In contrast to this, the number of unattached cornified envelopes on day 10 in the RT10 cells was increased by 85%, while the number of cornified, unattached 308 cells was similar to that in the controls. The effects of the pentapeptide thus seem to affect differentiation stronger than proliferation in the nontransformed cell line. Bivariate BrdUrd/DNA flow cytometry analysis on day 10 indicated that the reduced number of 308 cells was mainly due to a slower rate of cell proliferation and not to a increased sloughing off of keratinized cells. This analysis also demonstrated that an inhibition of DNA synthesis in the RT10 cells could be detected prior to a reduction of the cell number per well.     

Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells

Bone morphogenetic proteins (BMP) play a pivotal role in growth and differentiation of osteoblastic lineage cells. BMPs are potent stimulators of bone formation in various animal models. To understand the mechanism of BMP action in bone cells, we have investigated the effects of overexpression of the BMP-2 gene on proliferation and differentiation of UMR-106 rat osteosarcoma cells. A stable UMR-106 cell line overexpressing the BMP-2 gene was established by transfection of cells using a mammalian expression vector harboring human BMP-2 cDNA followed by G418 selection. After introduction of the BMP-2 gene, UMR-106 cells appeared more spindle-shaped in morphology compared to the predominantly cuboidal appearance of the parental cells. Overexpression of BMP-2 markedly inhibited proliferation as measured by cell counting and [3H]thymidine incorporation assays. Extracellular matrix (ECM) derived from cells overexpressing BMP-2 exhibited a less supportive effect on proliferation of UMR cells than did ECM derived from parental cells. Furthermore, cell-cell communication through gap junctions was reduced more than 50% as determined by nondisruptive fluorescent dye transfer assays. Overexpression of BMP-2 significantly stimulated expression of osteocalcin and alkaline phosphatase genes, indicating its role in osteoblastic differentiation. There was little effect on osteopontin gene expression.     

Oxidative stress modulates osteoblastic differentiation of vascular and bone cells

Oxidative stress may regulate cellular function in multiple pathological conditions, including atherosclerosis. One feature of the atherosclerotic plaque is calcium mineral deposition, which appears to result from the differentiation of vascular osteoblastic cells, calcifying vascular cells (CVC). To determine the role of oxidative stress in regulating the activity of CVC, we treated these cells with hydrogen peroxide (H(2)O(2)) or xanthine/xanthine oxidase (XXO) and assessed their effects on intracellular oxidative stress, differentiation, and mineralization. These agents increased intracellular oxidative stress as determined by 2,7 dichlorofluorescein fluorescence, and enhanced osteoblastic differentiation of vascular cells, based on alkaline phosphatase activity and mineralization. In contrast, H(2)O(2) and XXO resulted in inhibition of differentiation markers in bone osteoblastic cells, MC3T3-E1, and marrow stromal cells, M2-10B4, while increasing oxidative stress. In addition, minimally oxidized low-density lipoprotein (MM-LDL), previously shown to enhance vascular cell and inhibit bone cell differentiation, also increased intracellular oxidative stress in the three cell types. These effects of XXO and MM-LDL were counteracted by the antioxidants Trolox and pyrrolidinedithiocarbamate. These results suggest that oxidative stress modulates differentiation of vascular and bone cells oppositely, which may explain the parallel buildup and loss of calcification, seen in vascular calcification and osteoporosis, respectively.     

Nitric oxide modulates interleukin-2-induced proliferation in CTLL-2 cells

The role of the L-arginine-nitric oxide metabolic pathway was explored for interleukin-2-induced proliferation in the cytotoxic T lymphocyte clone CTLL-2. Specific inhibition of nitric oxide synthase significantly diminished, in a concentration-dependent manner, (3)H-thymidine uptake of CTLL-2 cells in response to different concentrations of interleukin 2. Withdrawal of L-arginine from culture medium resulted as potent as the higher inhibition obtained when blocking nitric oxide synthase with L-arginine analogues. Furthermore, intermedial concentrations of Larginine and exogenous nitric oxide donors were found for achieving optimal IL2-induced proliferation of CTLL-2. These findings prompted us to suggest that intra- and/or inter-cellular nitric oxide signalling may contribute to the modulation of the IL2 mitogenic effect upon cytotoxic T lymphocytes.     

The orphan receptor tyrosine kinase Ror2 modulates canonical Wnt signaling in osteoblastic cells

Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

Inhibitory effects of beta 2-microglobulin on in vitro calcification of osteoblastic cells

Elevated levels of aluminum and beta 2-microglobulin have been demonstrated in chronic dialysis patients. The role of aluminum in the pathogenesis of renal osteodystrophy has also been shown. We report on the effects of beta 2-microglobulin on calcification in vitro using osteoblastic cells, clone MC3T3-El. At concentrations comparable to those in plasma of chronic dialysis patients, both beta 2-microglobulin and aluminum suppressed calcification while collagen synthesis and alkaline phosphatase activity were maintained. These observations may be related to the impaired bone mineralization frequently observed in chronic dialysis patients.     

Vitamin K dependent in vitro production of prothrombin

During prothrombin biosynthesis, glutamyl residues in prothrombin precursor proteins are carboxylated to gamma-carboxyglutamyl residues by a vitamin K dependent carboxylase. Calcium-dependent and calcium-independent rat prothrombin antibody subpopulations have been produced and utilized to study the liver microsomal precursors of prothrombin that accumulate when vitamin K action is blocked. A substantial portion of the precursor pool accumulating in the vitamin K deficient or warfarin-treated rat will react with a Ca2+-dependent antibody at high calcium concentration and appears to be partially carboxylated. During in vitro incubation in the presence of vitamin K, the fraction of the precursor pool which is tightly bound to the microsomal membrane appears to be the preferred substrate for the vitamin K dependent carboxylation. A small amount of completely carboxylated rather than a large amount of partially carboxylated products are produced during these incubations. Treatment with a Sepharose-bound prothrombin antibody demonstrated that about 20-25% of the total carboxylated microsomal protein precursor pool consists of prothrombin precursors. This treatment removes an equal amount of total carboxylase activity, and the enzyme is active in this carboxylase precursor-antibody complex.     

Regulated expression of MCP-1 by osteoblastic cells in vitro and in vivo.

Inflammation is characterized by the recruitment of leukocytes from the vasculature. Recent studies have implicated chemokines as an important class of mediators that function principally to stimulate leukocyte recruitment, and in some cases, leukocyte activity. There are four defined chemokine subfamilies based on their primary structure, CXC, CC, C and CX3C. Members of the CC chemokine subfamily, such as monocyte chemoattractant protein 1 (MCP-1), are chemotactic for monocytes and other leukocyte subsets. The studies described below focus on the expression of MCP-1 in vitro and in vivo in an osseous environment. These studies indicate that MCP-1 is typically not expressed in normal bone or by normal osteoblasts in vitro. Upon stimulation by inflammatory mediators, MCP-1 is up-regulated. This expression is temporally and spatially associated with the recruitment of monocytes in both osseous inflammation and during developmentally regulated bone remodelling. Furthermore, exogenous MCP-1 applied to inflamed bone enhances the recruitment of monocytes. Because monocytes produce factors that influence osseous metabolism, including but not limited to prostglandins, platelet-derived growth factor, interleukin-1 or tumor necrosis factor, chemokines that initiate their recruitment are likely to be highly important.     

Musashi2 modulates K562 leukemic cell proliferation and apoptosis involving the MAPK pathway

The RNA-binding protein Musashi2 (Msi2) has been identified as a master regulator within a variety of stem cell populations via the regulation of translational gene expression. A recent study has suggested that Msi2 is strongly expressed in leukemic cells of acute myeloid leukemia patients, and elevated Msi2 is associated with poor prognosis. However, the potential role of Msi2 in leukemogenesis is still not well understood. Here, we investigated the effect of Msi2 knockdown on the biological properties of leukemic cells. High expression of Msi2 was found in K562 and KG-1a leukemic cell lines, and low expression was observed in the U937 cell line. We transduced K562 cells with two independent adenoviral shRNA vectors targeting Msi2 and confirmed knockdown of Msi2 at the mRNA and protein levels. Msi2 silencing inhibited cell growth and caused cell cycle arrest by increasing the expression of p21 and decreasing the expression of cyclin D1 and cdk2. In addition, knockdown of Msi2 promoted cellular apoptosis via the upregulation of Bax and downregulation of Bcl-2 expression. Furthermore, Msi2 knockdown resulted in the inactivation of the ERK/MAPK and p38/MAPK pathways, but no remarkable change in p-AKT was observed. These data provide evidence that Msi2 plays an important role in leukemogenesis involving the MAPK signaling pathway, which indicates that Msi2 may be a novel target for leukemia treatment.     

BMP-2 modulates the proliferation and differentiation of normal and cancerous gastric cells

Fibroblast growth factor (FGF) is established as an initiator of signaling events critical for neurogenesis and mesoderm formation during early Xenopus embryogenesis. However, less is known about the role FGF signaling plays in endoderm specification. Here, we show for the first time that endoderm-specific genes are induced when FGF signaling is blocked in animal cap explants. This block of FGF signaling is also responsible for a significant enhancement of endodermal gene expression in animal cap explants that are injected with a dominant-negative BMP-4 receptor (DNBR) RNA or treated with activin, however, neural and mesoderm gene expression is diminished. Consistent with these results, the injection of dominant-negative FGF receptor (DNFR) RNA expands endodermal cell fate boundaries while FGF treatment dramatically reduces endoderm in whole embryos. Taken together, these results indicate that inhibition of FGF signaling promotes endoderm formation, whereas the presence of active FGF signaling is necessary for neurogenesis/mesoderm formation.     

A proteomic study on human osteoblastic cells proliferation and differentiation

Changes in expression profiles for 17 proteins were ascertained in human mature osteoblasts compared to pre-osteoblasts (differentiation markers). A differential approach was used to highlight proteomic changes between human osteosarcoma cells and mature osteoblasts, showing a relative over-expression of 8 proteins (proliferation and tumor indicators), as well as under-expression of proteins also found down-regulated in pre-osteoblasts (specific markers of osteoblast differentiation). Our findings confirmed the differences between cell lines and primary human cell cultures and suggested caution on the use of osteosarcoma to study anti-osteoporotic drugs in humans.     

Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro

Menaquinone-7, which is vitamin K₂ (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10^–7–10^–5 M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10^–6 M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10^–7–10^–5 M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.