Michael Martin Ollmann

Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area

Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin‐concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), α‐melanin‐stimulating hormone (α‐MSH), and agouti‐related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual‐label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP‐ and NPY‐immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas α‐MSH‐immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY‐, AgRP‐, and α‐MSH‐immunoreactive fibers. Projections from populations of leptin‐responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight. J. Comp. Neurol. 402:442–459, 1998.

Modulation of murine melanocyte function in vitro by agouti signal protein

Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, α‐melanocyte‐stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan‐a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome‐like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time‐ and dose‐dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase‐related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.

THE ROLE OF AGOUTI-RELATED PROTEIN IN REGULATING BODY WEIGHT

Defects in signaling by leptin, a hormone produced primarily by adipose tissue that informs the brain of the bodys energy reserves, result in obesity in mice and humans. However, the majority of obese humans do not have abnormalities in leptin or its receptor but instead exhibit leptin resistance that could result from defects in downstream mediators of leptin action. Recently, two potential downstream mediators, agouti-related protein (Agrp) and its receptor, the melanocortin-4 receptor (Mc4r), have been identified. Agrp and Mc4r are excellent candidates for human disorders of body weight regulation and represent promising targets for pharmacological intervention in the treatment of these disorders.

Down-regulation of melanocortin receptor signaling mediated by the amino terminus of Agouti protein in Xenopus melanophores.

Agouti protein and Agouti-related protein (Agrp) regulate pigmentation and body weight, respectively, by antagonizing melanocortin receptor signaling. A carboxyl-terminal fragment of Agouti protein, Ser73-Cys131, is sufficient for melanocortin receptor antagonism, but Western blot analysis of skin extracts reveals that the electrophoretic mobility of native Agouti protein corresponds to the mature full-length form, His23-Cys131. To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopusmelanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time- and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein.

Molecular Pharmacology of Agouti Protein in Vitro and in Vivo

ABSTRACT: Agouti protein and Agouti‐related protein (Agrp) are paracrine signaling molecules that act by antagonizing the effects of melanocortins, and several alternatives have been proposed to explain their mechanisms of action. Genetic crosses in a sensitized background uncover a phenotypic difference between overexpression of Agouti and loss of Mc1r function, demonstrate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from α‐MSH stimulation. In vitro, Agouti protein inhibits melanocortin action by two mechanisms: competitive antagonism that depends on the carboxy‐terminus of the protein, and downregulation of melanocortin receptor signaling that depends on the aminoterminus. Our findings provide evidence of a novel signaling mechanism whereby α‐MSH and Agouti protein function as independent ligands that inhibit each others binding and transduce opposite signals through a single receptor.

Construction, analysis, and application of a radiation hybrid mapping panel surrounding the mouse agouti locus

The region surrounding the agouti coat color locus on mouse Chromosome 2 contains several genes required for peri-implantation development, limb morphogenesis, and segmentation of the nervous system. We have applied radiation hybrid mapping, a somatic cell genetic technique for constructing long-range maps of mammalian chromosomes, to eight molecular markers in this region. Using a mathematical model to estimate the frequency of radiation-induced breakage, we have constructed a map that spans approximately 20 recombination units and 475 centirays8000. The predicted order of markers, Prn-p-Pygb-Emv-13-Psp-Xmv-10-Emv-15-Src-Ada, is consistent with a previously derived multipoint meiotic map for six of the eight markers and suggests that Xmv-10 may lie relatively close to one or more of the agouti recessive lethal mutations. The resolution of our map is approximately 40-fold higher than the meiotic map, but the median retention frequency of mouse DNA in hybrid cells, 0.12, is 4-fold lower than similar experiments with human chromosomes. From one of the radiation hybrid lines that contained a minimum amount of mouse DNA, 25 independent cosmids were isolated with a mouse-specific hybridization probe. Single-copy fragments from two of these cosmids were shown to originate from mouse Chromosome 2, and the meiotic map position of one was found to be within 10 recombination units of the region of interest. Our results indicate more precise map positions for Pygb and Xmv-10, demonstrate that radiation hybrid mapping can provide high-resolution map information for the mouse genome, and establish a new method for isolating large fragments of DNA from a specific subchromosomal region.

Differential spontaneous transformation in vitro of newly established mouse fibroblast lines carrying or lacking the viable yellow mutation (Avy) of the mouse agouti locus

The pleiotropic effects of the viable yellow mutation (Avy), an allele of the mouse agouti coat‐color locus, include increased susceptibility to spontaneous and chemically induced tumors that affect a wide variety of tissues. As a first step toward understanding the molecular basis of this phenomenon, we established permanent fibroblast‐like cell lines from newborn Avy/a and control congenic a/a mice and compared their growth characteristics in vitro. From the VY/WffC3Hf/Nctr and YS/WffCH3f/Nctr‐Avy inbred strains, each of which carries the Avy allele on a congenic background, 38 clonal Avy/a and 16 clonal a/a lines were established. Regardless of inbred strain, all Avy/a cell lines exhibited a significant degree of spontaneous transformation, as assessed by focus formation in monolayer culture, whereas none of the a/a cell lines formed foci in prolonged cultures. To test whether changes in dosage of the Avy‐ or a‐bearing chromosomes were related to these events, we analyzed each cell line with a closely linked molecular probe from the Emv‐15 locus, which in the VY strain detects a restriction fragment length variant (RFLV) informative for the Avy‐ and a‐bearing chromosomes. Most of the transformed foci maintained heterozygosity for RFLVs detected by the probe, but two of the transformants lost the a‐associated RFLV, and at least one of the transformants exhibited amplification of the Avy‐associated RFLV. When the transformants were analyzed with 5′ sequences derived from the recently cloned agouti gene, three of eight transformants lost the a‐associated RFLV, and two of the transformants showed amplification of the Avy‐associated RFLV. Reverse transcriptase‐polymerase chain reaction assays indicated that agouti RNA was detected in Avy/a, not a/a cell lines. Surprisingly, some of the Avy/a transformants lacked agouti RNA. These results suggest that deregulated expression of the Avy allele is required for the initiation but not for the maintenance of transformation of the Avy/a cell cultures. These cell lines may provide an in vitro culture system for studying the effect of the agouti gene on tumorigenicity as well as to potentially study other pleiotropic phenotypes.

Association of Xmv-10 and the non-agouti (a) mutation explained by close linkage instead of causality

In a previous survey of endogenous proviruses among inbred mouse strains, the Xmv-10 provirus was found only in strains that carried the non-agouti (a) mutation (Frankel et al. J. Virol. 63: 1763–1774, 1989). To determine whether insertion of Xmv-10 caused the a mutation, we cloned a portion of Xmv-10 and its insertion site. Using a fragment of flanking cellular DNA as a Southern hybridization probe, we found that the Xmv-10 provirus was still present in revertant alleles of a to ator AW.A restriction fragment length variant (RFLV) in cellular DNA at the Xmv-10 insertion site was found to be correlated with the presence or absence of the provirus among inbred strains of laboratory mice regardless of their agouti allele. This correlation did not extend to wild mice, however, in which none of the samples contained Xmv-10, yet one, Mus domesticus poschiavinus, contained the insertion site RFLV correlated with Xmv-10 in laboratory mice. Analysis of an intersubspecific backcross with RFLVs at the insertion sites of Xmv-10 and Emv-15 (an endogenous provirus associated with Ay)revealed the following genetic map information: cen-A-0.31±0.31 cM-Emv-15-0.62±0.27 cM-Xmv-10-tel. Haplotype analysis of inbred strains in which a was not associated with Xmv-10 and in which Aywas not associated with Emv-15 demonstrated that these “exceptions” were explained most simply by a single recombination that disturbed the linkage relationships evident in most inbred strains. These results demonstrate that Xmv-10 did not cause the a mutation, suggest that insertion of Xmv-10 occurred recently in the evolution of laboratory mice, and show that the associations between agouti alleles and endogenous proviruses are due to linkage disequilibrium.

Abstract C192: Characterization of the target profile of XL228, a multi‐targeted protein kinase inhibitor in phase 1 clinical development

Background: XL228 is a small molecule protein kinase inhibitor currently in Phase 1 clinical development targeting Ph+ leukemias, multiple myeloma, and solid tumors. Cellular and in vivo data have demonstrated activity of XL228 against IGF1R, ABL (including the T315I variant) and SRC family kinases. XL228 was tested in a panel of biochemical protein kinase assays, and additional putative targets were identified, including Aurora family kinases, FGFR1‐3, and ALK. As these potential targets are known to enhance cell proliferation and survival in certain malignancies, the validation of their inhibition by XL228 through in vitro studies and in clinical pharmacodynamic samples was performed. Results: A panel of kinase inhibitors including XL228 was profiled against a series of cancer cell lines with known alterations in major signaling pathways. Approximately 30% of the lines demonstrated XL228 IC50 values of 4N DNA content, suggesting a failure of cytokinesis and consequent endoreduplication. Activity of XL228 against Aurora kinases was further verified by dose‐dependent inhibition of histone H3 phosphorylation, where it showed approximately 5‐fold higher potency than the control compound VX‐680. To confirm the relevance of the in vitro observations to the clinical activity of XL228, tumor biopsy specimens obtained from the solid tumor Phase 1 clinical study were evaluated for changes in phosphorylation of pathway markers by immunofluorescence. Inhibition of Aurora, FGFR, IGF1R, and SRC family kinases was observed, as measured by decreases in kinase substrate phosphorylation in these specimens. Conclusions: XL228 shows a broad pattern of protein kinase inhibition, including the tyrosine kinases IGF1R, SRC, ABL, FGFR1‐3, and ALK and the serine/threonine kinases Aurora A and Aurora B. The clinical impact of this target profile is currently being assessed in two Phase 1 studies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C192.