Showing papers in "Journal of Cellular Physiology in 2006"

Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates

Abstract: Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose-derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non-cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+ CD45- cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood-derived cells (CD45+), ASCs (CD31- CD34+ CD45- CD90+ CD105- CD146-), endothelial (progenitor) cells (CD31+ CD34+ CD45- CD90+ CD105low CD146+), pericytes (CD31- CD34- CD45- CD90+ CD105- CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10-20 weeks. These results suggest that liposuction-derived cells may be useful and valuable for cell-based therapies.

Leptin and cancer

Abstract: The prevalence of obesity has markedly increased over the past two decades, especially in the industrialized countries. While the impact of excess body weight on the development of cardiac disease and diabetes has been well documented, the link between obesity and carcinogenesis is just being recognized. This review will focus on the link between leptin, a cytokine that is elevated in obese individuals, and cancer development. First, we briefly discuss the biological functions of leptin and its signaling pathways. Then, we summarize the effects of leptin on different cancer types in experimental cellular and animal models. Next, we analyze epidemiological data on the relationship between obesity and the presence of cancer or cancer risk in patients. Finally, leptin as a target for cancer treatment and prevention will be discussed.

MT1-MMP: a potent modifier of pericellular microenvironment.

Abstract: Cells are regulated by many different means, and there is more and more evidence emerging that changes in the microenvironment greatly affect cell function. MT1-MMP is a type I transmembrane proteinase which participates in pericellular proteolysis of extracellular matrix (ECM) macromolecules. The enzyme is cellular collagenase essential for skeletal development, cancer invasion, growth, and angiogenesis. MT1-MMP promotes cell invasion and motility by pericellular ECM degradation, shedding of CD44 and syndecan1, and by activating ERK. Thus MT1-MMP is one of the factors that influence the cellular microenvironment and thereby affect cell-signaling pathways and eventually alters cellular behavior. As a proteinase, MT1-MMP is regulated by inhibitors, but it also requires formation of a homo-oligomer complex, localization to migration front of the cells, and internalization to become a "functionally active" cell function modifier. Developing new means to inhibit "functional activity" of MT1-MMP may be a new direction to establish treatments for the diseases that MT1-MMP mediates such as cancer and rheumatoid arthritis.

Clonal analysis of the differentiation potential of human adipose-derived adult stem cells

Abstract: Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF‐κB‐independent, FoxO1‐dependent mechanism

Abstract: Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.

Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase

Abstract: Competency for self-renewal of human embryonic stem (ES) cells is linked to pluripotency. However, there is a critical paucity of fundamental parameters of human ES cell division. In this study we show that human ES cells (H1 and H9; NIH-designated WA01 and WA09) rapidly proliferate due to a very short overall cell cycle (15–16 h) compared to somatic cells (e.g., normal diploid IMR90 fibroblasts and NT-2 teratocarcinoma cells). The human ES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened. Bromodeoxyuridine (BrdU) incorporation and FACS analysis demonstrated that 65% of asynchronously growing human ES cells are in S phase. Immunofluorescence microscopy studies detecting BrdU labeled mitotic chromosomes, Ki67 domains, and p220NPAT containing Cajal bodies revealed that the durations of the S (∼8 h), G2 (∼4 h), and M phases (∼1 h) are similar in ES and somatic cells. We determined that human ES cells remain viable after synchronization with either nocodazole or the anti-tumor drug Paclitaxel (taxol) and have an abbreviated G1 phase of only 2.5–3 h that is significantly shorter than in somatic cells. Molecular analyses using quantitative RT-PCR demonstrate that human ES cells and somatic cells express similar cell cycle markers. However, among cyclins and cyclin-dependent kinases (CDKs), we observed high mRNA levels for the G1-related CDK4 and cyclin D2 genes. We conclude that human ES cells exhibit unique G1 cell cycle kinetics and use CDK4/cyclin D2 related mechanisms to attain competency for DNA replication. J. Cell. Physiol. 209: 883–893, 2006. © 2006 Wiley-Liss, Inc.

Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs

Abstract: Low oxygen tension is thought to be an integral component of the human mesenchymal stem cell (hMSC) native bone marrow microenvironment. HMSC were cultured under physiologically relevant oxygen environments (2% O2) in three-dimensional (3D) constructs for up to 1 month in order to investigate the combined effects of chronic hypoxia and 3D architecture on hMSC tissue-development patterns. Hypoxic hMSC exhibited an extended lag phase in order to acclimatize to culture conditions. However, they subsequently proliferated continuously throughout the culture period, while maintaining significantly higher colony-forming unit capabilities and expressing higher levels of stem cell genes than hMSC cultured at 20% O2 (normoxic) conditions. Upon induction, hypoxic hMSC also expressed higher levels of osteoblastic and adipocytic differentiation markers than normoxic controls. Hypoxia induced increased total protein levels in hMSC throughout the culture period, as well as significantly different fibronectin expression patterns suggesting that oxygen levels can significantly affect tissue-development patterns. Importantly, hMSC maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSC niche. Further studies are required to determine how variations in cellular characteristics and ECM expression impact on the physiological properties of the engineered tissue, yet these results strongly indicate that oxygen tension is a key parameter that influences the in vitro characteristics of hMSC and their development into tissues.

Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells.

Abstract: The nuclear Ki-67 protein (pKi-67) has previously been shown to be exclusively expressed in proliferating cells. As a result, antibodies against this protein are widely used as prognostic tools in cancer diagnostics. Here we show, that despite the strong downregulation of pKi-67 expression in non-proliferating cells, the protein can nevertheless be detected at sites linked to ribosomal RNA (rRNA) synthesis. Although this finding does not argue against the use of pKi-67 as a proliferation marker, it has wide ranging implications for the elucidation of pKi-67 function. Employing the novel antibody TuBB-9, we could further demonstrate that also in proliferating cells, a fraction of pKi-67 is found at sites linked to the rRNA transcription machinery during interphase and mitosis. Moreover, chromatin immunoprecipitation (ChIP) assays provided evidence for a physical association of pKi-67 with chromatin of the promoter and transcribed region of the rRNA gene cluster. These data strongly suggest a role for pKi-67 in the early steps of rRNA synthesis. © 2005 Wiley-Liss, Inc.

Functions of human replication protein A (RPA) : From DNA replication to DNA damage and stress responses

Abstract: Human replication protein A (RPA), a heterotrimeric protein complex, was originally defined as a eukaryotic single-stranded DNA binding (SSB) protein essential for the in vitro replication of simian virus 40 (SV40) DNA. Since then RPA has been found to be an indispensable player in almost all DNA metabolic pathways such as, but not limited to, DNA replication, DNA repair, recombination, cell cycle, and DNA damage checkpoints. Defects in these cellular reactions may lead to genome instability and, thus, the diseases with a high potential to evolve into cancer. This extensive involvement of RPA in various cellular activities implies a potential modulatory role for RPA in cellular responses to genotoxic insults. In support, RPA is hyperphosphorylated upon DNA damage or replication stress by checkpoint kinases including ataxia telangiectasia mutated (ATM), ATR (ATM and Rad3-related), and DNA-dependent protein kinase (DNA-PK). The hyperphosphorylation may change the functions of RPA and, thus, the activities of individual pathways in which it is involved. Indeed, there is growing evidence that hyperphosphorylation alters RPA-DNA and RPA-protein interactions. In addition, recent advances in understanding the molecular basis of the stress-induced modulation of RPA functions demonstrate that RPA undergoes a subtle structural change upon hyperphosphorylation, revealing a structure-based modulatory mechanism. Furthermore, given the crucial roles of RPA in a broad range of cellular processes, targeting RPA to inhibit its specific functions, particularly in DNA replication and repair, may serve a valuable strategy for drug development towards better cancer treatment.

The p53 tumor suppressor participates in multiple cell cycle checkpoints.

Abstract: The process of cell division is highly ordered and regulated. Checkpoints exist to delay progression into the next cell cycle phase only when the previous step is fully completed. The ultimate goal is to guarantee that the two daughter cells inherit a complete and faithful copy of the genome. Checkpoints can become activated due to DNA damage, exogenous stress signals, defects during the replication of DNA, or failure of chromosomes to attach to the mitotic spindle. Abrogation of cell cycle checkpoints can result in death for a unicellular organism or uncontrolled proliferation and tumorigenesis in metazoans (Nyberg et al., 2002). The tumor suppressor p53 plays a critical role in each of these cell cycle checkpoints and is reviewed here.

The Cbl family proteins: ring leaders in regulation of cell signaling.

Abstract: The proto-oncogenic protein c-Cbl was discovered as the cellular form of v-Cbl, a retroviral transforming protein. This was followed over the years by important discoveries, which identified c-Cbl and other Cbl-family proteins as key players in several signaling pathways. c-Cbl has donned the role of a multivalent adaptor protein, capable of interacting with a plethora of proteins, and has been shown to positively influence certain biological processes. The identity of c-Cbl as an E3 ubiquitin ligase unveiled the existence of an important negative regulatory pathway involved in maintaining homeostasis in protein tyrosine kinase (PTK) signaling. Recent years have also seen the emergence of novel regulators of Cbl, which have provided further insights into the complexity of Cbl-influenced pathways. This review will endeavor to provide a summary of current studies focused on the effects of Cbl proteins on various biological processes and the mechanism of these effects. The major sections of the review are as follows: Structure and genomic organization of Cbl proteins; Phosphorylation of Cbl; Interactions of Cbl; Localization of Cbl; Mechanism of effects of Cbl: (a) Ubiquitylation-dependent events: This section elucidates the mechanism of Cbl-mediated downregulation of EGFR and details the PTK and non-PTKs targeted by Cbl. In addition, it addresses the functional requirements for E3 Ubiquitin ligase activity of Cbl and negative regulation of Cbl-mediated downregulation of PTKs, (b) Adaptor functions: This section discusses the mechanisms of adaptor functions of Cbl in mitogen-activated protein kinase (MAPK) activation, insulin signaling, regulation of Ras-related protein 1 (Rap1), PI-3' kinase signaling, and regulation of Rho-family GTPases and cytoskeleton; Biological functions: This section gives an account of the diverse biological functions of Cbl and includes the role of Cbl in transformation, T-cell signaling and thymus development, B-cell signaling, mast-cell degranulation, macrophage functions, bone development, neurite growth, platelet activation, muscle degeneration, and bacterial invasion; Conclusions and perspectives.

Long‐term cryopreservation of dental pulp stem cells (SBP‐DPSCs) and their differentiated osteoblasts: A cell source for tissue repair

Abstract: It is not known whether cells derived from stem cells retain their differentiation and morpho-functional properties after long-term cryopreservation. This information is of importance to evaluate their potential for long-term storage with a view to subsequent use in therapy. Here, we describe the morpho-functional properties of dental pulp stem cells (SBP-DPSCs), and of their differentiated osteoblasts, recovered after long-term cryopreservation. After storage for 2 years, we found that stem cells are still capable of differentiation, and that their differentiated cytotypes proliferate and produce woven bone tissue. In addition, cells still express all their respective surface antigens, confirming cellular integrity. In particular, SBP-DPSCs differentiated into pre-osteoblasts, showing diffuse positivity for ALP, BAP, RUNX-2, and calcein. Recovered osteoblasts expressed bone-specific markers and were easily recognizable ultrastructurally, with no alterations observed at this level. In addition, after in vivo transplantation, woven bone converted into a 3D lamellar bone type. Therefore, dental pulp stem cells and their osteoblast-derived cells can be long-term cryopreserved and may prove to be attractive for clinical applications.

An approachable human adult stem cell source for hard‐tissue engineering

Abstract: Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/CD34+/STRO-1+ cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10(-8) mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies.

Formation of elongated giant mitochondria in DFO-induced cellular senescence: involvement of enhanced fusion process through modulation of Fis1.

Abstract: Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H2O2 (200 µM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF β1- or H2O2-induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-ΔTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence. J. Cell. Physiol. 209: 468–480, 2006. © 2006 Wiley-Liss, Inc.

Differential subcellular distribution of glucose transporters GLUT1–6 and GLUT9 in human cancer: Ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

Abstract: It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2–6 and 9. The classical expression of GLUT1–5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers. J. Cell. Physiol. © 2006 Wiley-Liss, Inc.

Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells.

Abstract: The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant k(OFF) of individual GFP-labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques--chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices--increases the k(OFF) of the focal adhesion protein zyxin. In contrast, the k(OFF) of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force-dependent increases in zyxin's k(OFF) that occur over seconds are sufficient to quantitatively predict large-scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force-dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells.

Subcutaneous tissue fibroblast cytoskeletal remodeling induced by acupuncture: Evidence for a mechanotransduction-based mechanism

Abstract: Acupuncture needle rotation has been previously shown to cause specific mechanical stimulation of subcutaneous connective tissue. This study uses acupuncture to investigate the role of mechanotransduction-based mechanisms in mechanically-induced cytoskeletal remodeling. The effect of acupuncture needle rotation was quantified by morphometric analysis of mouse tissue explants imaged with confocal microscopy. Needle rotation induced extensive fibroblast spreading and lamellipodia formation within 30 min, measurable as an increased in cell body cross sectional area. The effect of rotation peaked with two needle revolutions and decreased with further increases in rotation. Significant effects of rotation were present throughout the tissue, indicating the presence of a response extending laterally over several centimeters. The effect of rotation with two needle revolutions was prevented by pharmacological inhibitors of actomyosin contractility (blebbistatin), Rho kinase (Y-27632 and H-1152), and Rac signaling. The active cytoskeletal response of fibroblasts demonstrated in this study constitutes an important step in understanding cellular mechanotransduction responses to externally applied mechanical stimuli in whole tissue, and supports a previously proposed model for the mechanism of acupuncture involving connective tissue mechanotransduction.

Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells

Abstract: Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed.

Wnt induction of chondrocyte hypertrophy through the Runx2 transcription factor.

Abstract: We investigated the molecular mechanisms underlying canonical Wnt-mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over-expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP-2)-induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF-beta-induced expression of Sox9 and col2a1. Over-expression of beta-catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and beta-catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical beta-catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of beta-catenin-TCF/Lef on Runx2 promoter. Co-transfection of lymphoid enhancer factor (Lef1) and beta-catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt-mediated induction of Runx2. Mutation of the TCF/Lef binding site in the -128 fragment of the Runx2 promoter resulted in loss of its responsiveness to beta-catenin. Additionally, gel-shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site-directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/beta-catenin signaling is regulated by TGF-beta and BMP-2 in chick upper sternal chondrocytes, and mediates chondrocyte hypertrophy at least partly through activation of Runx2 which in turn may induce col10a1 expression.

Survivin study: An update of “What is the next wave?”

Abstract: Studies on survivin over the past 2-3 years have shown that survivin possesses multiple subcellular localizations and is a multifunctional molecule involved in many aspects of cellular processes and/or behaviors. The subcellular localization and function of the survivin splice variants, however, have not yet been well elucidated. We have, therefore, provided additional observations on several survivin splice variants for further exploration. This review article will update the role of survivin, and its splice variants in the mitosis/cell cycle, apoptosis, tumorigenesis, chemoprevention, drug/radiation resistance, and cancer therapeutics.

Inhibition of human embryonic stem cell differentiation by mechanical strain

Abstract: Mechanical forces have been reported to induce proliferation and/or differentiation in many cell types, but the role of mechanotransduction during embryonic stem cell fate decisions is unknown. To ascertain the role of mechanical strain in human embryonic stem cell (hESC) differentiation, we measured the rate of hESC differentiation in the presence and absence of biaxial cyclic strain. Above a threshold of 10% cyclic strain, applied to a deformable elastic substratum upon which the hESC colonies were cultured, hESC differentiation was reduced and self-renewal was promoted without selecting against survival of differentiated or undifferentiated cells. Frequency of mechanical strain application had little effect on extent of differentiation. hESCs cultured under cyclic strain retained pluripotency, evidenced by their ability to differentiate to cell lineages in all three germ layers. Mechanical inhibition of hESC differentiation could not be traced to secretion of chemical factors into the media suggesting that mechanical forces may directly regulate hESC differentiation. Mechanical strain is not sufficient to inhibit differentiation, however, in unconditioned medium, hESCs grown under strain differentiated at the same rate as cells cultured in the absence of strain. Thus, while mechanical forces play a role in regulating hESC self-renewal and differentiation, they must act synergistically with chemical signals. These findings imply that application of mechanical forces may be useful, in combination with chemical and matrix-encoded signals, towards controlling differentiation of hESCs for therapeutic applications.

Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters.

Abstract: To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface-associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre-existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time-lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells. J. Cell. Physiol. 207: 87‐96, 2006. 2005 Wiley-Liss, Inc. Our understanding of the extracellular matrix (ECM) has expanded greatly over the past two decades. In addition to its well-known structural function, we now know that the ECM plays major biological roles in cellular adhesion, differentiation, and migration. A central function of the ECM, however, remains the creation and maintenance of tissue architecture and mechanical function. An underlying principle of matrix formation is the self-assembly of complex structures wherein monomeric units bind to each other to form oligomers and polymers. The information for proper assembly is encoded in the monomeric units themselves, yet there is increasing evidence that assembly of ECM structures is assisted directly by the cell (Yurchenco

Differentiation-related changes in mitochondrial properties as indicators of stem cell competence.

Abstract: Several methods may be used to assess stem cell competence, including the expression of cell surface markers and telomerase activity. We hypothesized that mitochondrial characteristics might be an additional and reliable way to verify stem cell competence. In a multipotent, adult monkey stromal stem cell line, previously shown to differentiate into adipocytes, chondrocytes, and osteocytes, we found that several mitochondrial properties change with increasing passage number in culture. Cells from the earliest passage (P11) versus those from a later passage (P17) are characterized by: (a) a much higher percentage of cells (85% vs. 18%) with a perinuclear arrangement of mitochondria; (b) a much lower percentage of cells (1% vs. 57%) with an aggregated mitochondrial arrangement, in which mitochondria appear to coalesce into large clumps; (c) a much lower percentage of cells with lipid droplets (1% vs. 36%), suggesting less differentiation into adipocytes; (d) a 5.6-fold lower ATP content per cell (0.45 vs. 2.51 pmoles ATP/cell; and (e) a 10-fold higher rate of oxygen consumption (37.8 vs. 3.8 nmoles O2/min/10(3) cells), indicating a higher metabolic activity. Collectively, these data indicate that the perinuclear arrangement of mitochondria, accompanied by a low ATP/cell content and a high rate of oxygen consumption, may be valid indicators of stem cell differentiation competence, while departures from this profile indicate that cells are differentiating or perhaps becoming senescent. These results represent the first characterization of mitochondrial properties reported for a primate stem cell line.

Steering estrogen signals from the plasma membrane to the nucleus: two sides of the coin.

Abstract: Estrogen mediate its biological effects through its association with estrogen receptors (ERs). They also regulate the expression of a variety of genes involved in distinct physiological processes, including development, metabolism, and reproduction. In addition, emerging data suggest that the estrogen-estrogen receptor complex can also function as a cytoplasmic signaling molecule and may influence processes such as cardiovascular protection, bone preservation, neuroprotection, and proliferation of various cell types. Such extranuclear or nongenomic signaling pathways are rapid and supposedly independent of transcription. A recent exciting finding was that G-coupled membrane protein receptor, GPR30, an alternative to the classical ERs, is also involved in the rapid signaling of estrogen through its direct association with estrogen. These new findings combined with the recent advances in the cytoplasmic functions of proline, glutamic acid, luecine rich protein 1 (PELP1), and metastatic tumor antigen 1 short form (MTA1s) have opened a new spectrum and raised several new concerns in the field of estrogen biology and put the attention to unveil many unknown mechanistic actions of estrogen in cellular physiology. In this review, we briefly summarize what is currently known of the cellular mechanisms and physiology of estrogen's nongenomic actions in various cellular systems used by ERs.

Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca2+ stimulates ERK, CREB, early genes.

Abstract: Controlled generation of reactive oxygen species (ROS) may contribute to physiological intracellular signaling events. We determined ROS generation in primary cultures of rat skeletal muscle after field stimulation (400 1-ms pulses at a frequency of 45 Hz) or after depolarization with 65 mM K+ for 1 min. Both protocols induced a long lasting increase in dichlorofluorescein fluorescence used as ROS indicator. Addition of diphenyleneiodonium (DPI), an inhibitor of NAD(P)H oxidase, PEG-catalase, a ROS scavenger, or nifedipine, an inhibitor of the skeletal muscle voltage sensor, significantly reduced this increase. Myotubes contained both the p47phox and gp91phox phagocytic NAD(P)H oxidase subunits, as revealed by immunodetection. To study the effects of ROS, myotubes were exposed to hydrogen peroxide (H2O2) at concentrations (100–200 µM) that did not alter cell viability; H2O2 induced a transient intracellular Ca2+ rise, measured as fluo-3 fluorescence. Minutes after Ca2+ signal initiation, an increase in ERK1/2 and CREB phosphorylation and of mRNA for the early genes c-fos and c-jun was detected. Inhibition of ryanodine receptor (RyR) decreased all effects induced by H2O2 and NAD(P)H oxidase inhibitors DPI and apocynin decreased ryanodine-sensitive calcium signals. Activity-dependent ROS generation is likely to be involved in regulation of calcium-controlled intracellular signaling pathways in muscle cells. J. Cell. Physiol. 209: 379–388, 2006. © 2006 Wiley-Liss, Inc.

Biological functions of maspin.

Abstract: Maspin (Mammary Serine Protease Inhibitor) was first reported in 1994 as a serpin with tumor suppressive properties. Maspin was initially isolated through subtractive hybridization and differential display analysis as a 42-kDa protein that is expressed in normal mammary epithelial cells but reduced or absent in breast carcinomas (Zou et al., 1994). Further research led to maspin's characterization as a class II tumor suppressor based on its ability to inhibit cell invasion, promote apoptosis, and inhibit angiogenesis (Sheng et al., 1996; Zhang et al., 2000b; Jiang et al., 2002). Since then, efforts have been made to characterize maspin's tumor suppressive mechanisms. In particular, researchers have studied maspin localization, the regulation of maspin expression, and more recently, maspin protein interactions. By elucidating these mechanisms, researchers are beginning to understand the complex, pleiotropic nature of maspin and the pathways through which maspin exerts its tumor suppressive properties. These new findings not only further enhance our understanding of cancer biology but also provide an avenue to develop maspin's potential as a diagnostic marker for cancer progression, and as a potentially powerful therapeutic agent in the fight against breast cancer.

Stochastic cancer progression driven by non-clonal chromosome aberrations.

Abstract: Cancer research has previously focused on the identification of specific genes and pathways responsible for cancer initiation and progression based on the prevailing viewpoint that cancer is caused by a stepwise accumulation of genetic aberrations This viewpoint, however, is not consistent with the clinical finding that tumors display high levels of genetic heterogeneity and distinctive karyotypes We show that chromosomal instability primarily generates stochastic karyotypic changes leading to the random progression of cancer This was accomplished by tracing karyotypic patterns of individual cells that contained either defective genes responsible for genome integrity or were challenged by onco-proteins or carcinogens that destabilized the genome Analysis included the tracing of patterns of karyotypic evolution during different stages of cellular immortalization This study revealed that non-clonal chromosomal aberrations (NCCAs) (both aneuploidy and structural aberrations) and not recurrent clonal chromosomal aberrations (CCAs) are directly linked to genomic instability and karyotypic evolution Discovery of "transitional CCAs" during in vitro immortalization clearly demonstrates that karyotypic evolution in solid tumors is not a continuous process NCCAs and their dynamic interplay with CCAs create infinite genomic combinations leading to clonal diversity necessary for cancer cell evolution The karyotypic chaos observed within the cell crisis stage prior to establishment of the immortalization further supports the ultimate importance of genetic aberrations at the karyotypic or genome level Therefore, genomic instability generated NCCAs are a key driving force in cancer progression The dynamic relationship between NCCAs and CCAs provides a mechanism underlying chromosomal based cancer evolution and could have broad clinical applications

Potentiation of TRPV3 channel function by unsaturated fatty acids.

Abstract: Transient receptor potential (TRP) channels have emerged as cellular sensors of physical and chemical changes inside and outside cells (Clapham, 2003). Six TRP channels have been shown to be involved in temperature sensing in sensory neurons and skin. TRPA1 and TRPM8 are involved in detecting cool to cold temperatures while TRPV1, V2, V3, V4 are responsible for sensing warm to noxious heat. In addition to thermosensation, these channels also respond to inflammatory mediators, implicating their involvement in inflammatory pain and tissue injury-induced thermal hyperalgesia. For example, TRPV1 is activated by bradykinin, nerve growth factor, and ATP through signaling pathways mediated by activation of their respective receptors (Cesare et al., 1999; Chuang et al., 2001; Tominaga et al., 2001) and by tissue acidosis as a consequence of inflammation and malignant tumor growth (Reeh and Kress, 2001). Hypotonicity-induced activation of TRPV4 in primary afferent nociceptive nerve fibers is enhanced by prostaglandin E2 (Alessandri-Haber et al., 2003). TRPA1 is activated by bradykinin through activation of phospholipase C (Bandell et al., 2004). In the inflamed tissues, arachidonic acid (AA) is either released from the infiltrating lymphocytes or produced within the sensory fibers or skin cells following the activation of receptors by other inflammatory mediators. While the lipoxygenase products of AA directly activate TRPV1 (Hwang et al., 2000; Shin et al., 2002), the epoxygenase products are responsible for the stimulatory effect of AA on TRPV4 (Watanabe et al., 2003). TRPV3 may be involved in the sensation of warm to noxious heat. The reported temperature threshold values for TRPV3 ranged from 31 to 39°C (Peier et al., 2002; Smith et al., 2002; Xu et al., 2002) and the channel was continuously activated up to 50°C (Xu et al., 2002). Expression of TRPV3 protein has been shown in mouse skin keratinocytes (Chung et al., 2003, 2004a) and in sensory neurons of human dorsal root ganglia (Smith et al., 2002). Knockout of TRPV3 gene from mice led to impaired responses to innocuous and noxious heat, which are believed to be due to a defect in thermasensation in the skin cells (Moqrich et al., 2005). Recently, we, and others, showed that TRPV3 channels are activated by 2-aminoethoxydiphenyl borate (2APB) (Hu et al., 2004; Chung et al., 2004b). With the use of 2APB, TRPV3-mediated heat-sensitive currents were detected in primary keratinocytes isolated from TRPV4−/− mice (Chung et al., 2004b). Because of the sequence homology and functional similarities between TRPV3 and other heat-sensitive channels, it has been speculated that TRPV3 also plays a role in thermal hypersensitivity induced by tissue injury and inflammation (Smith et al., 2002). Here we show that TRPV3 channel function is greatly enhanced by AA via a mechanism that does not require the oxidation of the fatty acid and the effect can be mimicked by other unsaturated fatty acids, including non-metabolizable analogues of AA. The potentiation by fatty acids is not mediated through protein kinase C (PKC) even though activation of PKC also causes an increase of TRPV3 activity. Parts of the results have appeared in an abstract form (Hu et al., 2005).

The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib

Abstract: We investigated the role of the MEK/MAPK pathway in the sensitivity/resistance of breast carcinoma cells to the EGFR tyrosine kinase inhibitor gefitinib (IRESSA). We assessed the effects of gefitinib on the growth of three breast cancer cell lines that showed high (SK-Br-3; IC50 4 microM), intermediate (MDA-MB-361; IC50 5.3 microM), and low (MDA-MB-468; IC50 6.8 microM) sensitivity to the drug. Although treatment with gefitinib inhibited EGFR activation in the three cell lines in a similar fashion, significant reduction of both p42/p44-MAPK and AKT phosphorylation was observed in SK-Br-3 and MDA-MB-361, but not in MDA-MB-468 cells. The growth of MDA-MB-468 cells was significantly inhibited by treatment with either the PI3K-inhibitor LY294002 or the MEK-inhibitor PD98059. In agreement with these findings, treatment of MDA-MB-468 cells with a combination of PD98059 and gefitinib produced a synergistic anti-tumor effect, whereas this combination was only additive in SK-Br-3 and MDA-MB-361 cells. The combination of gefitinib and PD98059 also produced a significant increase in the levels of apoptosis in MDA-MB-468 cells as compared with treatment with a single agent. This phenomenon was associated with a profound decrease in MAPK activation, reduction of BAD (ser112) phosphorylation and a paradoxical increase in the levels of AKT activation. Finally, overexpression of a constitutively activated form of p42-MAPK in MCF-10A non-transformed human mammary epithelial cells resulted in a two- to three-fold increase in the IC50 to gefitinib. Taken together, these data strongly support the role of the MEK/MAPK pathway in the resistance to gefitinib, and provide the rationale for novel therapeutic approaches based on combinations of signal transduction inhibitors.

Extracellular matrix deposition by fibroblasts is necessary to promote capillary-like tube formation in vitro.

Abstract: The contribution of the cellular and fibrillar microenvironment to angiogenesis still remains unclear. Our purpose was to evaluate the effect of the extracellular matrix deposited by fibroblasts on the capacity of human endothelial cells to form capillaries in vitro. We have drastically decreased the amount of extracellular matrix surrounding fibroblasts in our model of endothelialized-reconstructed connective tissue (ERCT) by culturing it without ascorbate. Under these conditions, the number of capillary-like tubes (CLT) formed by endothelial cells was reduced by up to 10-fold after 31 days of culture compared to controls. This decrease was due neither to a variation of MMP-2 and MMP-9 secretion, nor to a reduction in the number of fibroblasts and/or endothelial cells, or a diminution of fibroblast growth factor 2 (FGF2) synthesis. The secretion of vascular endothelial growth factor (VEGF) by fibroblasts accounted for 25-70% of the capillary-like tube formation when tissues were cultured in the presence or absence of ascorbate, as demonstrated by VEGF-blocking studies. The culture of endothelial cells on a similar extracellular matrix but in the absence of living fibroblasts did not promote the formation of CLT, even when tissues were fed with fibroblast-conditioned medium. Thus, the deposition of a rich extracellular matrix by living fibroblasts appeared necessary, but not sufficient to promote capillary-like formation. Fibroblasts seem to induce endothelial cells to spontaneously form CLT by secreting and organizing an abundant extracellular matrix, which creates a microenvironment around cells that could in turn trap growth factors produced by fibroblasts and promote three-dimensional cell organization.