In order to complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multi-dimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most a 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using CHO genome exclusively which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. 504 of the detected proteins included N-acetylation modifications and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.

Both intercellular signaling and epigenetic mechanisms regulate embryonic development, but it is unclear how they are integrated to establish and maintain lineage-specific gene expression programs. Here, we show that a key function of the developmentally essential Nodal-Smads2/3 (Smad2 and Smad3) signaling pathway is to recruit the histone demethylase Jmjd3 to target genes, thereby counteracting repression by Polycomb. Smads2/3 bound to Jmjd3 and recruited it to chromatin in a manner that was dependent on active Nodal signaling. Knockdown of Jmjd3 alone substantially reduced Nodal target gene expression, whereas in the absence of Polycomb, target loci were expressed independently of Nodal signaling. These data establish a role for Polycomb in imposing a dependency on Nodal signaling for the expression of target genes and reveal how developmental signaling integrates with epigenetic processes to control gene expression.

Nodal, a member of the transforming growth factor ␤ (TGF-␤) superfamily, is implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Here we define the intracellular signaling pathway induced by recombinant nodal protein treatment of P19 embryonal carcinoma cells. Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-␤. However, nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. We also demonstrate that nodal induces p(CAGA) 12 , a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduces the level of luciferase reporter activity induced by nodal treatment. Finally, we show that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide the first direct biochemical evidence that nodal signaling is mediated by both activin-TGF-␤ pathway Smads, Smad2 and Smad3. We also show here that the extracellular cripto protein is required for nodal signaling, making it distinct from activin or TGF-␤ signaling. Members of the transforming growth factor ␤ (TGF-␤)1 superfamily of intercellular signaling factors regulate cell fate and behavior during development and in the adult (1). The three major subgroups based on sequence similarity are the TGF-␤s, activins and inhibins, and bone morphogenetic proteins (BMPs; Ref. 1). Nodal and related factors form a separate subgroup and are implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification (2).Signaling by TGF-␤ and related ligands uses two types of receptors, type I and type II transmembrane serine-threonine kinases. Ligand binding results in the formation of heteromeric receptor complexes, in which type II receptors phosphorylate type I receptors (1, 3). Downstream signal transduction events are mediated by the intracellular Smad proteins. One class, the receptor-regulated Smads (R-Smads), are directly phosphorylated by activated type I receptors on a C-terminal SSXS motif. Upon phosphorylation, R-Smads form complexes with the coSmad, Smad4 and then translocate to the nucleus and regulate transcription of target genes. Biochemical and biological studies have established that the R-Smads used by TGF-␤ and activin signaling, Smad2 and Smad3, are distinct from those used by BMP signaling, Smad1, Smad5, and Smad8 (1, 3, 4).The nodal signaling pathway awaits characterization at the biochemical level. However, mutational studies in the mouse and zebrafish and ectopic expression studies in Xenopus and zebrafish suggest that the nodal and activin signaling pathways may share receptors and Smads. Targeted mutations in the mouse Smad2 gene (5-8) and the activin type IB receptor gene (9) and combined mutations of the activin type IIA and IIB receptor genes (10) show gastrulation phenotypes resem...

The inflammatory cytokines interleukin-1␤ (IL-1␤Elevated plasma levels of cytokines, including IL-1␤ and TNF found in various inflammatory, infectious, and malignant diseases, are often associated with hypocholesterolemia (7-11). Systemic infusion of cytokines has been shown to lower serum cholesterol levels in animals and humans (12-14). The concentrations of IL-1␤ and TNF similar to those found following infection induce low density lipoprotein (LDL) receptor expression in human hepatoma (HepG2) cells, and this effect is not part of its mitogenic response, as IL-1␤ did not increase DNA synthesis (15-18). Interestingly, a 1.56-kilobase pair region of the 5Ј-flanking region of the human LDL receptor promoter has been shown to confer IL-1␤-dependent induction to an heterologous gene, suggesting that increased receptor expression results from activation of LDL receptor transcription and is not due to an alteration in LDL receptor mRNA stability (18). These studies have also established that, unlike TNF, IL-1␤-induced LDL receptor transcription does not require protein synthesis and, therefore, likely depends on activation of a preexisting component(s). The initial step in their action is the association of agonist with its cell surface receptors, followed by intracellular protein phosphorylation/dephosphorylation (19 -21). The downstream effectors linking receptor activation with the different cellular responses are still largely to be defined, although several signaling pathways have been proposed (20,21). The signal transduction mechanisms by which these cytokines stimulates LDL receptor expression are poorly characterized. Many extracellular signals elicit specific biological responses through activation of MAPK cascades (22-26). The three major subfamilies of MAPKs in higher eukaryotes include ERK-1/2, 46/54 JNK , and p38 MAPK , all of which are activated by phosphorylation of a tyrosine and a threonine residue catalyzed by a dual specificity MAPK kinase. ERKs are most strongly activated by mitogenic signals such as growth factors or 12-Otetradecanoylphorbol-13-acetate (TPA), whereas p46/54 JNK , and p38MAPK are activated by stressful stimuli such as the inflammatory cytokines, IL-1␤, and TNF, thermal shock, and osmotic shock (23,27). Activation of MAPKs leads to distinct cellular responses mediated by phosphorylation of specific target substrates (25). Recently, we have shown involvement of ERK-1/2 signaling cascade in TPA-induced LDL receptor expression in HepG2 cells (28).In this study, we have investigated the early in vivo signal-

The forkhead rhabdomyosarcoma transcription factor (FKHR) is a promising candidate to be the transcription factor that binds to the insulin response element of the insulin-like growth factor-binding protein-1 (IGF-BP-1) promoter and mediates insulin inhibition of IGF-BP-1 promoter activity. Cotransfection of mouse FKHR increased IGFBP-1 promoter activity 2-3-fold in H4IIE rat hepatoma cells; insulin inhibited FKHR-stimulated promoter activity ϳ70%. A C-terminal fragment of mouse FKHR (residues 208 -652) that contains the transcription activation domain fused to a Gal4 DNA binding domain potently stimulated Gal4 promoter activity. Insulin inhibited FKHR fragment-stimulated promoter activity by ϳ70%. Inhibition was abolished by coincubation with the phosphatidylinositol-3 kinase inhibitor, LY294002. The FKHR 208 -652 fragment contains two consensus sites for phosphorylation by protein kinase B (PKB)/Akt, Ser-253 and Ser-316. Neither site is required for insulin inhibition of promoter activity stimulated by the FKHR fragment, and overexpression of Akt does not inhibit FKHR fragment-stimulated Gal4 promoter activity. These results suggest that insulin-and phosphatidylinositol-3 kinase-dependent phosphorylation of another site in the fragment by a kinase different from PKB/Akt inhibits transcription activation by the fragment. Phosphorylation of this site also may be involved in insulin inhibition of transcription activation by full-length FKHR, but only after phosphorylation of Ser-253 by PKB/Akt.

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Insulin-like growth factor binding protein-3 (IGFBP-3) can inhibit cell growth by directly interacting with cells, as well as by forming complexes with IGF-I and IGF-II that prevent their growth-promoting activity. The present study examines the mechanism of inhibition of DNA synthesis by IGFBP-3 in CCL64 mink lung epithelial cells. DNA synthesis was measured by the incorporation of 5-bromo-2'-deoxyuridine, using an immunocolorimetric assay. Recombinant human IGFBP-3 (rh[N109D,N172D]IGFBP-3) inhibited DNA synthesis in proliferating and quiescent CCL64 cells. Inhibition was abolished by co-incubation of IGFBP-3 with a 20% molar excess of Leu(60)-IGF-I, a biologically inactive IGF-I analogue that binds to IGFBP-3 but not to IGF-I receptors. DNA synthesis was not inhibited by incubation with a preformed 1:1 molar complex of Leu(60)-IGF-I and IGFBP-3, indicating that only free IGFBP-3 inhibits CCL64 DNA synthesis. Inhibition by IGFBP-3 is not due to the formation of biologically inactive complexes with free IGF, since endogenous IGFs could not be detected in CCL64 conditioned media; any IGFs that might have been present could only have existed in inactive complexes, since endogenous IGFBPs were present in excess; and biologically active IGFs were not displaced from endogenous IGFBP complexes by Leu(60)-IGF-I. After incubation with CCL64 cells, (125)I-IGFBP-3 was covalently cross-linked to a major thick similar400-kDa complex. This complex co-migrated with a complex formed after incubation with (125)I-labeled transforming growth factor-beta (TGF-beta) that has been designated the type V TGF-beta receptor. (125)I-IGFBP-3 binding to the thick similar400-kDa receptor was inhibited by co-incubation with unlabeled IGF-I or Leu(60)-IGF-I. The ability of Leu(60)-IGF-I to decrease both the inhibition of DNA synthesis by IGFBP-3 and IGFBP-3 binding to the thick similar400-kDa receptor is consistent with the hypothesis that the thick similar400-kDa IGFBP-3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP-3.

Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC–MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO–CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.