Sheila M. Nielsen-Preiss

The role of annexin 2 in osteoblastic mineralization

While the basic cellular contributions to bone differentiation and mineralization are widely accepted, the regulation of these processes at the intracellular level remains inadequately understood. Our laboratory recently identified annexin 2 as a protein involved in osteoblastic mineralization. Annexin 2 was overexpressed twofold in SaOSLM2 osteoblastic cells as a fusion protein with green fluorescent protein. The overexpression of annexin 2 led to an increase in alkaline phosphatase activity as well as an increase in mineralization. Our data suggest that the increase in alkaline phosphatase activity does not result from increased alkaline phosphatase transcript or protein levels; therefore we evaluated mechanism of action. We determined that both annexin 2 and alkaline phosphatase activity were localized to membrane microdomains called lipid rafts in osteoblastic cells. Annexin 2 overexpression resulted in an increase in alkaline phosphatase activity that was associated with lipid microdomains in a cholesterol-dependent manner. Furthermore, disruption of lipid rafts with a cholesterol sequestering agent or reduction of annexin 2 expression by specific antisense oligonucleotides each resulted in diminished mineralization. Therefore, intact lipid rafts containing annexin 2 appear to be important for alkaline phosphatase activity and may facilitate the osteoblastic mineralization process.

Axl and Tyro3 Modulate Female Reproduction by Influencing Gonadotropin-Releasing Hormone Neuron Survival and Migration

GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl(-/-), Tyro3(-/-) double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.

Annexin 2 expression is reduced in human osteosarcoma metastases

Osteosarcoma is an aggressive primary bone cancer affecting primarily children and young adults. The development of valuable diagnostic indicators and therapeutic agents will be enhanced by the identification and characterization of genes that contribute to its aggressive behavior. We used representational difference analysis to isolate genes differentially expressed between primary human osteosarcoma tumors and subsequent metastatic lung lesions to identify genes potentially involved in metastatic potential. Several genes were differentially expressed between the two tumor populations, including annexin2. The levels of annexin2 mRNA and protein inversely correlated with metastatic potential in a subset of human osteosarcoma tumor specimens, as well as in a human osteosarcoma cell line selected for increased metastatic potential. Annexin2 has been described in several cellular localizations with various functional implications, many of which may be relevant to metastatic potential. Therefore, the subcellular localization of endogenous annexin2 protein was evaluated biochemically by subcellular fractionation and immunologically by flow cytometry and immunofluorescence in osteoblastic cells. Annexin2 was localized to the cytoplasm and intracellular aspect of the plasma membrane, excluded from the nucleus and undetectable on the cell surface or in the conditioned medium. Overexpression of annexin2 in osteosarcoma cells did not alter several in vitro phenotypes often used to assess metastatic potential including motility, adhesion, and proliferation. However, our previous data have implicated annexin2 in the mineralization process of osteoblastic cells in vitro. Consistent with an increase in differentiation‐induced mineralization, there was diminished tumorigenicity and experimental metastatic potential of osteosarcoma cells overexpressing annexin2. These data suggest that annexin2 may downregulate osteosarcoma aggressiveness by inducing a more differentiated state in osteoblastic cells.

Increased Filamentous Growth of Candida albicans in Simulated Microgravity

Knowledge of simulated microgravity (SMG)-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeast-hyphal transition. The morphological response may have significant implications for astronauts’ safety, as the fungal pathogen may become more virulent during spaceflight.

Spaceflight enhances cell aggregation and random budding in Candida albicans.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans–induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

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.

Establishment and characterization of OS 99-1, a cell line derived from a highly aggressive primary human osteosarcoma

Osteosarcoma is the most common form of primary bone cancer. In this study, we established a human osteosarcoma cell line (OS 99-1) from a highly aggressive primary tumor. G-banding karyotype analysis demonstrated a large number of clonal abnormalities, as well as extensive intercellular heterogeneity. Through the use of immunologic, molecular, and biochemical analyses, we characterized protein and gene expression profiles confirming the osteogenic nature of the cells. Further evaluation indicated that OS 99-1 cells maintain the capacity to differentiate in an in vitro mineralization assay as well as form tumors in the in vivo chicken embryo model. This cell line provides a useful tool to investigate the molecular mechanisms contributing to osteosarcoma and may have the potential to serve as a culture system for studies involving bone physiology.

Cancer stem cells: Seeds of growth in osteosarcoma

Commentary to: Characterization of stem cell attributes in human osteosarcoma cell lines Ling Wang, Paul Park, Chia-Yin Lin

118 DIFFERENTIAL EXPRESSION OF BONE MORPHOGENETIC PROTEIN-5, PROMYELOCYTIC LEUKEMIA ZINC FINGER AND MEMBERS OF THE GATA FAMILY IN GONADOTROPINOMAS

Gonadotropinomas, which produce α, LHβ, and/or FSHβ subunits, represent 35% of pituitary tumors. They pose a diagnostic challenge, presenting predominantly in men with headaches, decreased libido and impotence. Little is known of their pathogenesis, and identification of tumor-specific genes may help elucidate the molecular mechanisms that are involved. We used the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array to compare gene expression profiles in five gonadotropinomas with six pituitaries obtained from autopsies. Analysis with the Affymetrix GeneChip Operating System showed that 1549 of 39,000 genes examined were differentially expressed. We then used RT-PCR to analyze further several genes, which have been implicated in proliferation, that had unique expression patterns in our array. Bone morphogenetic proteins (BMPs) are growth factors involved in many aspects of tissue development and morphogenesis. Recently, members of the BMP family have been implicated in the production of FSH by gonadotropes and have been detected in gonadotropinomas. In contrast to prior literature, our microarray analysis showed that BMP-5 was selectively overexpressed 4.8-fold in gonadotropinomas compared with normal pituitary, which was confirmed by semi-quantitative RT-PCR. Thus, BMP-5 may have a previously undescribed role in FSH production and/or in gonadotrope proliferation. The promyelocytic leukemia zinc finger (PLZF) protein belongs to the POZ/zinc-finger family of transcription factors and acts as a negative regulator for GATA-2. GATA-2 and GATA-3 are found in pituitary cell lines and can regulate α-subunit gene expression, and GATA-2 has been detected in pituitary tumors. In our study, PLZF was underexpressed 13.9-fold in gonadotropinomas compared with normals, whereas GATA-2 and GATA-3 were overexpressed 6.7-fold and 26.5-fold, respectively. These data suggest that loss of the transcriptional repressor PLZF, together with activation of GATA family members, may be involved in the development of gonadotropinomas. Thus, various growth and transcription factors are differentially expressed in gonadotropinomas. The evaluation of candidate genes that emerge from these experiments provides a rational approach to investigate mechanisms underlying pituitary tumorigenesis.

221 IDENTIFICATION OF NOVEL FACTORS THAT REGULATE PITUITARY TUMORIGENESIS

Gonadotropinomas that make α, LHβ and/or FSHβ glycoprotein hormone subunits are the most prevalent type of pituitary macroadenoma. These tumors present a diagnostic challenge, as they are rarely associated with a characteristic clinical hypersecretory syndrome and thus difficult to recognize until large enough to cause mass effects. Mechanisms underlying the pathogenesis of gonadotropinomas have not been identified. Common oncogene mutations involved in other human cancers, such as Ras and Rb, are not involved in pituitary tumor formation. The aims of the current study were to elucidate molecular events responsible for the development of gonadotropinomas. We compared gene expression profiles between six normal human pituitaries and five glycoprotein-secreting tumors by cDNA microarray analysis. Pairwise comparisons of normal pituitaries and gonadotropinomas demonstrated consistent, greater than two-fold increased expression in 671 genes and decreased expression in 878 genes. Gene Ontology Database analysis was used to categorize differentially expressed genes into classes that may affect tumorigenic or cell proliferative properties. Relative to tumor versus normal, the following data were obtained: transcription factors (184 up/165 down), cell cycle regulation (104 up/62 down), apoptosis regulation (68 up/50 down), cancer genes (72 up/55 down), and signal transduction (158 up/240 down). Candidate genes were then selected based on the magnitude and consistency of change. RT-PCR was performed to confirm array-based results of differential expression in candidate genes. Deleted in Bladder Cancer Chromosome Region 1-Like (DBCCR1-L) is an attractive candidate as it was completely absent in all normal pituitary samples, but expressed in all tumor samples. DBCCR1-L is a novel gene. Its homologue, DBCCR1, is involved in G1 cell cycle transition and colon, breast, esophageal, ovarian, and bladder cancer. Investigation is underway to evaluate the consequences of DBCCR1-L knockdown and overexpression in pituitary cell lines and its effects on cell cycle progression. Our results demonstrate the differential expression of many genes, including DBCCR1-L, which may be involved in pituitary tumorigenesis. Future studies on candidate genes identified in this analysis may lead to the identification of tumor markers to detect, prognostically evaluate, and eventually medically treat gonadotropinomas. Sponsored by VA Merit to MEW and NIH and UCHSC Cancer Center Fellowships to DSM and MML