The low density lipoprotein receptor-related protein (LRP) 1 is a large endocytic receptor containing a 515-kDa heavy chain to which ligands bind and a non-covalently associated 85-kDa light chain containing a transmembrane and cytoplasmic domain (for review see Ref. 1). LRP is one of 12 or more receptors that make up the LDL receptor superfamily and is essential for embryonic development in mice (2). A remarkable feature of LRP is its ability to bind and mediate the internalization of a diverse array of ligands, including proteinases (3, 4), proteinase-inhibitor complexes (5, 6), and lipoproteins (7). After binding to the LRP, the ligands are transported into endosomes where they uncouple in the reduced pH environment and are sorted to lysosomes for degradation. LRP recycles back to the cell surface where it is once again available to bind ligands.Recent studies indicate that in addition to their cargo transport function, certain LDL receptor family members also participate in signaling pathways. For example, the very low density lipoprotein receptor and apoE receptor 2 both participate in a signal transduction pathways mediated by reelin (8 -10). Reelin is secreted by Cajal-Retzius cell in the outermost layer of the cerebral cortex and controls the final position of neurons that migrate from the ventricular zone. Binding of reelin to either the very low density lipoprotein receptor or apoE receptor 2 induces tyrosine phosphorylation of disabled-1 (Dab1) (9, 10), an adaptor protein that interacts with the cytoplasmic domains of LDL receptor family members (11, 12) and functions in tyrosine kinase signaling pathways.In the case of LRP, accumulating evidence suggests a prominent but undefined role for this receptor in regulating cell physiology by facilitating signal transduction pathways. For example, LRP has been implicated as a component of the receptor complex for midkine (13), a heparin binding growth factor with migration-promoting and survival-promoting activities. Another LRP ligand, tissue type plasminogen activator, promotes late phase long term potentiation (14), and this activity appears to require its association with LRP (15). Finally, the binding of activated ␣ 2 M (␣ 2 M*) to LRP mediates calcium influx in neurons in a process that also involves N-methyl-D-

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.

Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-a, designated mGRO-a, or KC. We have examined the possible role of mGRO-a expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 de®cient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-a and malignant potential with a pActin-KC vector encoding mGRO-a enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in in®ltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 de®cient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-a can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway.

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tive gene expression intensities, ratios or both are measured in order to quantify the gene expression level relative to its reference sample. Our early reports discussed a systematic data extraction algorithm in which a unique method of extracting gene expression intensities and ratios along with an adaptive ratio confidence interval, measurement qualities of gene expression ratios and intensities were presented. In many methods of gene expression data analysis, only expression ratios or normalized intensities are employed because of insufficient assessment at the individual data points (clones). Common practice dictates that data derived with poor measurement quality-such as expression ratios derived from weak reference expression levels or noise-corrupted measurements-shall not be used in the analysis. We present an automatic decision-making process for various algorithms, such as gene expression clustering and classification, in which gene expression ratios and intensities are chosen to participate in the analysis according to their measurement quality, expression signal-to-noise ratio relative to the reference channel and other parameters derived from cDNA microarray image analysis software. Chen, Zhong[36] To identify changes in gene expression associated with tumor progression and metastasis in vivo, we investigated differential gene expression in a metastatic squamous carcinoma model established in syngeneic mice, including a tumorigenic line PAM 212, and metastatic sublines derived from PAM 212 tumors, using mRNA differential display (DD) and cDNA microarrays. Using DD, seventy-two candidate cDNAs were detected, and thirty-four cDNAs were confirmed to be differentially expressed by northern blotting analysis. Global mRNA expression profiles were generated using an NCI mouse Oncochip composed of four thousand elements representing known genes and ESTs, plus 57 of the candidate cDNAs detected by DD to facilitate data validation. Clustering analysis of array results from metastatic cell lines and tumors identified a subset of genes that exhibited increased expression in the metastases, revealed that 22 unique clones are highly homologous to previously identified genes, and nine novel cDNAs. Strikingly, 10/22 of the genes identified have been associated with activation of the Nuclear Factor-kB signal transduction pathway. One of the genes identified, Gro-1, was recently confirmed to promote tumor growth, metastasis and angiogenesis of SCC in vivo in a separate report. These results demonstrate that early response pathway components and down stream genes related to NF-κB are expressed with metastatic tumor progression. Functional genomic approaches may promote a better understanding of the repertoire of related genes and molecular pathways involved in tumor progression and metastasis. Chodosh, Lewis[37] Functional analysis of mammary development using oligonucleotide microarraysStephen Master, Jennifer Hartman, Alexander Stoddard, Elizabeth Keiper, Susan Moody, Celina D'Cruz & Lewis Chodosh University of Pen...

Activation of the platelet-derived growth factor receptor-␤ (PDGFR-␤) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR-␤ was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-␤, we confirmed that the kinase domain of PDGFR-␤ is absolutely required for LRP tyrosine phosphorylation but that PDGFR-␤-mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-␥, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR-␤ may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR-␤ coimmunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR-␤ and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.The low density lipoprotein receptor-related protein (LRP) 1 is a large endocytic receptor composed of a 515-kDa heavy chain to which ligands bind and a non-covalently associated 85-kDa light chain containing a transmembrane and cytoplasmic domain. LRP is a member of the LDL receptor family and is required for embryonic development in mice (1). Although LRP was originally identified as an endocytic receptor for ␣-2-macroglobulin-protease complexes (1, 2) and apoE-enriched lipoprotein particles (3, 4), this receptor is now known to recognize more than 30 distinct ligands including lipoproteins, proteases, proteinase inhibitor complexes, matrix proteins, bacterial toxins, viruses, intracellular proteins, and growth factors (for a review, see Ref. 5).In addition to its prominent role in mediating the cellular uptake of a variety of ligands, recent work has implicated LRP in several signal transduction pathways and has expanded its realm to include regulation of cell migration (6 -8) and modulation of the integrity of the blood brain barrier (9). In the vasculature, LRP is expressed in smooth muscle cells, and a tissue-specific deletion of the LRP ge...

A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion coreceptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses. Adhesion of cells to the extracellular matrix (ECM)2 regulates a wide range of cellular processes, including cell survival, growth, migration, and differentiation. A central paradigm in the field entails both physical association and functional collaboration between integrins and growth factor receptors (GFRs) in the regulation of cell responses to the ECM and soluble growth factors (1). In particular, the engagement of ␤1 and ␣v␤3 integrins with ECM ligands transiently activates plateletderived growth factor (PDGF) receptor-tyrosine kinase even in

Human and murine squamous cell carcinomas (SCC) have been reported to produce proinflammatory cytokines IL-1alpha, IL-6, GM-CSF, and IL-8 or KC. Production of individual members of the proinflammatory cytokine family has been associated with increased tumor growth or metastasis in a variety of neoplasms. In this study, we determined whether the expression of these cytokines occurs as a result of the events of cellular transformation or culture, or is promoted by interaction of neoplastic cells with factors or cells in the host environment. We compared the expression of proinflammatory cytokines following the spontaneous transformation of murine keratinocytes in vitro, and following the formation of tumors and metastases from these transformed keratinocytes in syngeneic recipients in vivo. Using sensitive ELISA assays, we found that cultures of the in vitro transformed Balb/c SCC line Pam 212 do not produce elevated levels of proinflammatory cytokines IL-1alpha, IL-6, GM-CSF and KC, indicating that transformation or culture alone is insufficient to account for the level of cytokine expression detected in patient and experimental tumors. In contrast, Pam reisolates from primary and metastatic tumors were obtained which constitutively produce markedly elevated levels of cytokines IL-1alpha, IL-6, KC and GM-CSF. The increase in the expression of these cytokines by SCC in vivo occurred independent of T and B lymphocyte-mediated immunity, since increases in expression of the cytokines was observed in lines reisolated from immunodeficient athymic nude and SCID Balb/c congenic mice. The increased expression of cytokines appeared to result from additional events in vivo, rather than due to selection of a pre-existing cytokine-producing subpopulation, since clones of the parental cell line expressed lower cytokine levels than cloned reisolates, and clones of the non-secreting parental cell line that formed tumors in vivo secreted elevated levels of cytokines following reisolation. We conclude that the development of SCC that express proinflammatory cytokines is promoted by tumor-host interaction(s) that are independent of specific T and B cell immunity.