Jolene J. Windle

Immortalization of hypothalamic GnRH by genetically targeted tumorigenesis

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.

Establishment of an osteocyte-like cell line, MLO-Y4

Although osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T‐antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen‐coated surfaces which are optimal for osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO‐Y4 (for murine long bone osteocyte Y4). The properties of MLO‐Y4 cells are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline‐phosphatase. The cells produce extensive, complex dendritic processes and are positive for T‐antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO‐Y4 cells lack detectable mRNA for osteoblast‐specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on osteocyte function and for determining the means whereby osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.

Paget disease of bone

Paget disease of bone (PD) is characterized by excessive bone resorption in focal areas followed by abundant new bone formation, with eventual replacement of the normal bone marrow by vascular and fibrous tissue. The etiology of PD is not well understood, but one PD-linked gene and several other susceptibility loci have been identified, and paramyxoviral gene products have been detected in pagetic osteoclasts. In this review, the pathophysiology of PD and evidence for both a genetic and a viral etiology for PD will be discussed.

A Farnesyltransferase Inhibitor Induces Tumor Regression in Transgenic Mice Harboring Multiple Oncogenic Mutations by Mediating Alterations in Both Cell Cycle Control and Apoptosis

ABSTRACT The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)–v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV–v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV–v-Ha-ras mice were interbred with p53−/− mice. Tumors inras/p53−/− mice treated with L-744,832 regressed as efficiently as MMTV–v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV–v-Ha-ras mice were also interbred with MMTV–c-myc mice to determine whetherras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV–v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/mycmice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV–c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

Role of LPA4/p2y9/GPR23 in negative regulation of cell motility.

Lysophosphatidic acid (LPA) is a ligand of multiple G protein-coupled receptors. The LPA(1-3) receptors are members of the endothelial cell differentiation gene (Edg) family. LPA(4)/p2y9/GPR23, a member of the purinergic receptor family, and recently identified LPA(5)/GPR92 and p2y5 are structurally distant from the canonical Edg LPA receptors. Here we report targeted disruption of lpa(4) in mice. Although LPA(4)-deficient mice displayed no apparent abnormalities, LPA(4)-deficient mouse embryonic fibroblasts (MEFs) were hypersensitive to LPA-induced cell migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase pathway by LPA(4), LPA(4) deficiency potentiated Akt and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA(4) converted LPA(4)-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA(4) strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA(1) in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA(4) attenuated LPA(1)-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA(4) is a suppressor of LPA-dependent cell migration and invasion in contrast to the motility-stimulating Edg LPA receptors.

Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease

Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62(P392L) mutation contributes to the development of this disease. To determine the effects of p62(P392L) expression on osteoclasts in vitro and in vivo, we introduced either the p62(P392L) or WT p62 gene into normal osteoclast precursors and targeted p62(P392L) expression to the osteoclast lineage in transgenic mice. p62(P392L)-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-kappaB ligand (RANKL) and TNF-alpha and showed increased NF-kappaB signaling but did not demonstrate increased 1,25-(OH)(2)D(3) responsivity, TAF(II)-17 expression, or nuclear number per osteoclast. Mice expressing p62(P392L) developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62(P392L) expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.

Tissue-specific gene expression in the pituitary: the glycoprotein hormone alpha-subunit gene is regulated by a gonadotrope-specific protein.

The molecular mechanisms for the development of multiple distinct endocrine cell types in the anterior pituitary have been an area of intensive investigation. Though the homeodomain protein Pit-1/GHF-1 is known to be involved in differentiation of the somatotrope and lactotrope lineages, which produce growth hormone and prolactin, respectively, little is known of the transcriptional regulators important for the gonadotrope cell lineage, which produces the glycoprotein hormones luteinizing hormone and follicle-stimulating hormone. Using transgenic mice and transfection into a novel gonadotrope lineage cell line, we have identified a regulatory element that confers gonadotrope-specific expression to the glycoprotein hormone alpha-subunit gene. A tissue-specific factor that binds to this element is purified and characterized as a 54-kDa protein which is present uniquely in cells of the gonadotrope lineage and is not Pit-1/GHF-1. The human and equine alpha-subunit genes are also expressed in placental cells. However, the previously characterized placental transcription factors designated TSEB and alpha-ACT are not found in the pituitary gonadotrope cells, indicating that independent mechanisms confer expression of these genes in the two different tissues.

Osteoclasts are important for bone angiogenesis

Increased osteoclastogenesis and angiogenesis occur in physiologic and pathologic conditions. However, it is unclear if or how these processes are linked. To test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dose-dependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with parathyroid hormone related protein (PTHrP) increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with receptor activator of nuclear factor-kappaB ligand (RANKL) or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined whether matrix metalloproteinase-9 (MMP-9), an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo, respectively, from Mmp9(-/-) mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most probably by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9.

Increased tumor proliferation and genomic instability without decreased apoptosis in MMTV-ras mice deficient in p53.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53(-/-) mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53(+/+), ras/p53(+/-), or ras/p53(-/-)). ras/p53(-/-) mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53(+/+) and ras/p53(+/-) mice were of mammary origin, ras/p53(-/-) mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53(-/-) mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53(+/+) mice. Interestingly, the increased growth rates of ras/p53(-/-) tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.

Expression of Measles Virus Nucleocapsid Protein in Osteoclasts Induces Paget's Disease‐Like Bone Lesions in Mice

We targeted the MVNP gene to the OCL lineage in transgenic mice. These mice developed abnormal OCLs and bone lesions similar to those found in Pagets patients. These results show that persistent expression of MVNP in OCLs can induce pagetic‐like bone lesions in vivo.