Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Summary Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10–25mm glucose, alanine or leucine. Resistances of inter-cellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01–10 kHz. The segments were then fixedin situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixedin situ at 38°C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

Denatonium as a bitter taste receptor agonist damages jejunal epithelial cells of yellow-feathered chickens via inducing apoptosis

The sense of bitter taste is critical for chickens to acquire and select feeds. It is important to understand the roles and mechanisms of bitter taste transduction in chickens. Denatonium is extensively used as a bitter taste receptor agonist to activate bitter taste receptors in recent studies. The objective of this study was to investigate the physiological effects and the potential molecular mechanisms of dietary exposure to a strong bitter taste receptor agonist on the jejunal epithelial cells of yellow-feathered chickens. A total of 240 yellow-feathered chickens were divided into four treatments receiving a normal diet (Control), a low-dose denatonium treatment (Control + 5 mg/kg denatonium), a middle-dose denatonium treatment (Control + 20 mg/kg denatonium) and a high-dose denatonium treatment (Control + 100 mg/kg denatonium) for 56 days, respectively. The results showed that dietary denatonium reduced (P < 0.05) the growth performance of chickens. High-dose denatonium damaged the morphology of the jejunal epithelium and decreased (P < 0.05) the activities of Ca²⁺-ATPase, sucrase and maltase after 56 days of exposure. Meanwhile, high-dose denatonium increased (P < 0.05) mRNA expressions of bitter taste receptors, which resulted in enhanced apoptosis in jejunal epithelial cells after 56 days of exposure. Furthermore, middle-dose and high-dose denatonium exhibited increased (P < 0.05) mRNA level of claudin 2 and decreased (P < 0.05) mRNA level of occludin after 28 days of exposure. Only high-dose denatonium decreased (P < 0.05) mRNA level of occludin after 56 days of exposure. In conclusion, denatonium manifested deleterious effects on the jejunum of chickens in a dose–effect manner via damaging the morphology of the jejunal epithelium, and inducing apoptosis associated with bitter taste receptors. Our data suggest that bitter-tasting feed additives may have side effects on the growth and development of intestines in chickens.     

Inflammatory cytokine regulation of TRAIL-mediated apoptosis in thyroid epithelial cells

Death receptor-mediated apoptosis has been implicated in target organ destruction in chronic autoimmune thyroiditis. Depending on the circumstances, inflammatory cytokines such as IL-1, TNF and IFNgamma have been shown to contribute to either the induction, progression or inhibition of this disease. Here we demonstrate that the death ligand TRAIL can induce apoptosis in primary, normal, thyroid epithelial cells under physiologically relevant conditions, specifically, treatment with the combination of inflammatory cytokines IL-1beta and TNFalpha. In contrast, IFNgamma is capable of blocking TRAIL-induced apoptosis in these cells. This regulation of TRAIL-mediated apoptosis by inflammatory cytokines appears to be due to alterations of cell surface expression of TRAIL receptor DR5 and not DR4. We also show the in vivo presence of TRAIL and TRAIL receptors DR5 and DcR1 in both normal and inflamed thyroids. Our data suggests TRAIL-mediated apoptosis may contribute to target organ destruction in chronic autoimmune thyroiditis.     

Structure of the N-terminal half of gelsolin bound to actin: roles in severing, apoptosis and FAF

The actin filament-severing functionality of gelsolin resides in its N-terminal three domains (G1-G3). We have determined the structure of this fragment in complex with an actin monomer. The structure reveals the dramatic domain rearrangements that activate G1-G3, which include the replacement of interdomain interactions observed in the inactive, calcium-free protein by new contacts to actin, and by a novel G2-G3 interface. Together, these conformational changes are critical for actin filament severing, and we suggest that their absence leads to the disease Finnish-type familial amyloidosis. Furthermore, we propose that association with actin drives the calcium-independent activation of isolated G1-G3 during apoptosis, and that a similar mechanism operates to activate native gelsolin at micromolar levels of calcium. This is the first structure of a filament-binding protein bound to actin and it sets stringent, high-resolution limitations on the arrangement of actin protomers within the filament.     

Nuclear BAG-1 expression inhibits apoptosis in colorectal adenoma-derived epithelial cells

BAG-1 is an anti-apoptotic protein that is frequently deregulated in a variety of malignancies including colorectal cancer. There are three isoforms: BAG-1L is located in the nucleus, BAG-1M and BAG-1S are located both in the nucleus and the cytoplasm. In colon cancer, the expression of nuclear BAG-1 is associated with poorer prognosis and is potentially a useful predictive factor for distant metastasis. However, the function of BAG-1 in colonic epithelial cells has not been studied. Having previously shown a predominant nuclear localisation of BAG-1 in adenoma-derived cell lines,¹ we wanted to determine the function of nuclear BAG-1 in these non-tumourigenic cells, to identify whether nuclear BAG-1 was implicated in tumour progression in the colon. In the current report we established that nuclear BAG-1 inhibits apoptosis in a colorectal adenoma-derived cell line. We demonstrate that apoptosis induced by -radiation or the vitamin D analogue EB1089 in the non-tumourigenic human colorectal adenoma-derived S/RG/C2 cell line, was preceded by a decrease in nuclear and an increase in cytoplasmic BAG-1 expression. This change in subcellular localisation of BAG-1 was due to the redistribution of the BAG-1M isoform. In addition, we have shown that the maintenance of high nuclear BAG-1 through enforced expression of the nuclear localised BAG-1L isoform enhanced cellular survival after -radiation or exposure to EB1089. Furthermore the expression of cytoplasmic BAG-1S isoform fused with a nuclear localisation signal protected against -radiation induced apoptosis. This demonstrates that nuclear localisation of the BAG-1 protein confers a survival advantage in colorectal adenoma-derived cells and that nuclear BAG-1 could potentially be an important survival factor in colorectal carcinogenesis.     

Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells

Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto's thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-gamma and IL-1beta cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-gamma/IL-1beta pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.     

A novel microRNA mmu-miR-466h affects apoptosis regulation in mammalian cells

This study determined the changes in microRNA (miRs) expression in mammalian Chinese hamster ovary (CHO) cells undergoing apoptosis induced by exposing the cells to nutrient-depleted media. The apoptosis onset was confirmed by reduced cell viability and Caspase-3/7 activation. Microarray comparison of known mouse and rat miRs in CHO cells exposed to fresh or depleted media revealed up-regulation of the mouse miR-297-669 cluster in CHO cells subjected to depleted media. The mmu-miR-466h was chosen for further analysis as the member of this cluster with the highest overexpression and its up-regulation in depleted media was confirmed with qRT-PCR. Since miRs suppress mRNA translation, we hypothesized that up-regulated mmu-miR-466h inhibits anti-apoptotic genes and induces apoptosis. A combination of bioinformatics and experimental tools was used to predict and verify mmu-miR-466h anti-apoptotic targets. 8708 predicted targets were obtained from miRecords database and narrowed to 38 anti-apoptotic genes with DAVID NCBI annotation tool. Several genes were selected from this anti-apoptotic subset based on nucleotide pairing complimentarity between the mmu-miR-466h seed region and 3' UTR of the target mRNAs. The qRT-PCR analysis revealed reduced mRNA levels of bcl2l2, dad1, birc6, stat5a, and smo genes in CHO cells exposed to depleted media. The inhibition of the mmu-miR-466h increased the expression levels of those genes and resulted in increased cell viability and decreased Caspase-3/7 activation. The up-regulation of mmu-miR-466h in response to nutrients depletion causes the inhibition of several anti-apoptotic genes in unison. This suggests the pro-apoptotic role of mmu-miR-466h and its capability to modulate the apoptotic pathway in mammalian cells.     

冠状病毒感染调控细胞凋亡机制研究进展

冠状病毒是常见的感染人类和动物并造成健康危害的主要病原性微生物之一,冠状病毒感染细胞后,细胞产生免疫应答,病毒为了在细胞内转录翻译和装配下一代,应对细胞免疫应答的同时,还参与到许多细胞活动中,当细胞特定受体与病毒蛋白结合后,细胞即启动凋亡程序。冠状病毒的许多蛋白在细胞凋亡程序中起促进或抑制凋亡的不同作用,如病毒S蛋白与细胞膜死亡受体作用诱导细胞启动外在凋亡途径,病毒感染细胞后产生的M、S蛋白引起细胞内质网应激、Ca2+失衡,诱导细胞启动内在凋亡途径,而E蛋白则抑制细胞凋亡的发生。本文综述了冠状病毒对侵染细胞的促凋亡或抑制凋亡作用及其作用机制,通过了解病毒不同蛋白在各种凋亡途径中的不同作用,希望为人工干预调控细胞研究提供思路,为冠状病毒感染防控提供理论支持。     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.     

Nucleolar reorganization in epithelial cells of the jejunum of the rat

Light microscope studies were made on nucleoli of jejunal epithelial cells of normal rats fixed in OsO₄, glutaraldehyde, F.A.A. and HgCl₂ and stained with basic fuchsin–alkaline methylene blue. Nucleolar reorganization is extensive and clearly resembles the phenomenon of nucleolar segregation. Polymorphous nucleoli of undifferentiated crypt cells show intermingled constituents and stain purple whereas similar nucleoli of definitive absorptive cells show two homogeneous components–A, stained red and B, stained blue. Cytochemical studies indicate that component A is largely protein and acidophilic and component B is largely nucleic acids and basophilic. These nucleoli become compacted, each forming an amphinucleolus with the two components at opposite poles. Further changes occur along the villi and the components generally separate to form a condensed plasmosome and a diffuse karysome. Extruded cells show nucleolar fragmentation. Electron micrographs of OsO₄ material were used in preparation of wax models. These, along with electron micrographs of glutaraldehyde material stained with uranyl acetate and lead citrate, clearly illustrate and duplicate light microscope findings and strongly resemble nucleolar segregation produced by antimetabolites. Cells of the villi with reorganized nucleoli do not undergo mitosis whereas undifferentiated crypt cells do so. Furthermore, nucleolar reorganization is correlated with aging since it begins in crypt cells and culminates in senescent cells at the tips of the villi. A review is given of the extensive evidence showing that, in the intestine certain functional changes occur similar to those demonstrated in experimental nucleolar segregation. These include gradually changing patterns of DNA, RNA and protein synthesis as well as enzyme activity. The accompany and probably result from nucleolar reorganization.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

缺血预适应对大鼠肢体缺血/再灌注后小肠细胞凋亡的影响

目的：观察大鼠肢体缺血再灌注后小肠粘膜自由基及钙含量改变与细胞凋亡情况．以及缺血预适应对其变化的影响。方法：将雄性Wistar大鼠18只,随机分为对照（Control）组,缺血／再灌注（I／R）组和缺血预适应（IPC＋I／R）组,分别测定血浆和小肠组织超氧化物歧化酶（SOD）、黄嘌呤氧化酶（XOD）、丙二醛（MDA）的含量,小肠组织钙及线粒体钙含量;小肠组织的Bel-2和Bax蛋白的表达水平;检测小肠细胞凋亡情况。结果：肢体I／R后血浆和小肠粘膜SOD减少而XOD和MDA增加;小肠组织钙及线粒体钙含量增多;Bel-2蛋白表迭和Bax表达增多,但Bel-2／Bax比值降低;凋亡细胞增多。IPC减轻了I／R后引起的XOD、MDA含量的升高,并且增加了SOD的含量;减轻了组织和线粒体钙超载;Bel-2的表达则明显升高而Bax表达较I／R组明显减少,Bel-2／Bax比值升高;凋亡细胞减少。结论：肢体IR引起小肠粘膜自由基的增多,钙超栽,凋亡细胞增多;IPC可能通过减少自由基的产生及钙超载,抑制细胞凋亡而对肢体I／R继发的小肠功能损伤起保护作用。     

持续性细胞皱缩在人上皮细胞凋亡过程中的必要性

持续性细胞皱缩是凋亡发生的一个主要标志。近期研究发现细胞皱缩在细胞凋亡过程中并不是一个被动的次要事件。在各种细胞中,包括人上皮细胞,凋亡因子（apoptogen）刺激后马上发生全细胞皱缩,又称为凋亡性容积减小（apoptotic　volumede　crease,AVD）,继而发生caspase激活、DNA片段化、细胞破裂死亡。K^＋和Cl^-通道的激活导致KCl外流,诱导AVD发生。抑制AVD发生可以抑制细胞凋亡。AVD与调节性容积增加（regulatory　volume　increase,RVI）异常相伴发生时,人上皮性HeLa细胞发生持续性细胞皱缩。RVI功能受损时,高渗本身就能诱导HeLa细胞持续性细胞皱缩,继而凋亡。即使在正常渗透压、无凋亡因子刺激的情况下,将HeLa细胞置于缺乏Na^＋或Cl。的溶液也会导致细胞持续性皱缩,继而凋亡。因此,AVD诱导和RVI异常所导致的持续性细胞皱缩是人上皮细胞发生凋亡的首要条件。     

The molecular regulation of organelle transport in mammalian retinal pigment epithelial cells

Retinal pigment epithelial cells contain large numbers of melanosomes that can enter the apical processes extending between the outer segments of the overlying photoreceptors. Every day the distal portion of the photoreceptor outer segment is shed and phagocytosed by the retinal pigment epithelial cell. The phagosome is then transported into the cell body and the contents degraded by lysosomal enzymes. This review focuses on recent progress made in the identification of molecules that regulate the transport of melanosomes into the apical processes and the transport of phagosomes into the cell body. Myosin VIIa is a key player in both processes and, at least in the case of melanosome movement, myosin VIIa is recruited to the melanosome via the GTPase, Rab27a. The possible role played by defects in the transport of melanosomes and phagosomes in the development of retinal degenerative diseases is discussed.     

EGFR plays a pivotal role in the regulation of polyamine-dependent apoptosis in intestinal epithelial cells

Intracellular polyamine synthesis is regulated by the enzyme ornithine decarboxylase (ODC), and its inhibition by -difluromethylornithine (DFMO), confers resistance to apoptosis. We have previously shown that DFMO leads to the inhibition of de novo polyamine synthesis, which in turn rapidly activates Src, STAT3 and NF-κB via integrin β3 in intestinal epithelial cells. One mechanism to explain these effects involves the activation of upstream growth factor receptors, such as the epidermal growth factor receptor (EGFR). We therefore hypothesized that EGFR phosphorylation regulates the early response to polyamine depletion. DFMO increased EGFR phosphorylation on tyrosine residues 1173 (pY1173) and 845 (pY845) within 5 min. Phosphorylation declined after 10 min and was prevented by the addition of exogenous putrescine to DFMO containing medium. Phosphorylation of EGFR was concomitant with the activation of ERK1/2. Pretreatment with either DFMO or EGF for 1 h protected cells from TNF-/CHX-induced apoptosis. Exogenous addition of polyamines prevented the protective effect of DFMO. In addition, inhibition of integrin β3 activity (with RGDS), Src activity (with PP2), or EGFR kinase activity (with AG1478), increased basal apoptosis and prevented protection conferred by either DFMO or EGF. Polyamine depletion failed to protect B82L fibroblasts lacking the EGFR (PRN) and PRN cells expressing either a kinase dead EGFR (K721A) or an EGFR (Y845F) mutant lacking the Src phosphorylation site. Conversely, expression of WT-EGFR (WT) restored the protective effect of polyamine depletion. Fibronectin activated the EGFR, Src, ERKs and protected cells from apoptosis. Taken together, our data indicate an essential role of EGFR kinase activity in MEK/ERK-mediated protection, which synergizes with integrin β3 leading to Src-mediated protective responses in polyamine depleted cells.     

ABCG2 expression and uric acid metabolism of the intestine in hyperuricemia model rat

Abstract

To elucidate roles of the intestine in uric acid (UA) metabolism, we examined ABCG2 expression, tissue UA content and xanthine oxidoreductase (XOR) activity in different intestinal segments. Male SD rats were assigned to control group or oxonic acid-induced hyperuricemia (HUA) group. In control rats, ABCG2 was present both in villi and crypts in each segment. Tissue UA content and XOR activity were relatively high in duodenum and jejunum. However, in HUA rats, tissue UA content was significantly elevated in the ileum, whereas it remained unaltered in other segments. Moreover, ABCG2 expression in the HUA group was upregulated both in the villi and crypts of the ileum. These data indicate that the ileum may play an important role in the extra-renal UA excretion.     

Role of PTEN and Akt in the regulation of growth and apoptosis in human osteoblastic cells

Cancer cells are characterized by either an increased ability to proliferate or a diminished capacity to undergo programmed cell death. PTEN is instrumental in regulating the balance between growth and death in several cell types and has been described as a tumor suppressor. The chromosome arm on which PTEN is located is deleted in a subset of human osteosarcoma tumors. Therefore, we predicted that the loss of PTEN expression was contributing to increased Akt activation and the subsequent growth and survival of osteosarcoma tumor cells. Immunoblot analyses of several human osteosarcoma cell lines and normal osteoblasts revealed relatively abundant levels of PTEN. Furthermore, stimulation of cell growth or induction of apoptosis in osteosarcoma cells failed to affect PTEN expression or activity. Therefore, routine regulation of osteosarcoma cell growth and survival appears to be independent of changes in PTEN. Subsequently, the activation of a downstream target of PTEN activity, the survival factor Akt, was analyzed. Inappropriate activation of Akt could bypass the negative regulation by PTEN. Analyses of Akt expression in several osteosarcoma cell lines and normal osteoblasts revealed uniformly low basal levels of phosphorylated Akt. The levels of phosphorylated Akt did not increase following growth stimulation. In addition, osteosarcoma cell growth was unaffected by inhibitors of phosphatidylinositol-3 kinase, an upstream activator of the Akt signaling pathway. These data further suggest that the Akt pathway is not the predominant signaling cascade required for osteoblastic growth. However, inhibition of PTEN activity resulted in increased levels of Akt phosphorylation and enhanced cell proliferation. These data suggest that while abundant levels of PTEN normally maintain Akt in an inactive form in osteoblastic cells, the Akt signaling pathway is intact and functional.