Control of therapeutic IgG antibodies galactosylation during cultivation process and its impact on IgG1/FcγR interaction and ADCC activity.

Therapeutic monoclonal antibodies (mAbs) are within the fastest growing group of pharmaceuticals on the global market. IgG1 subclass is the most potent effector in Fc-related functions. The N-linked glycosylation of mAbs Fc-domain significantly influences its therapeutic activity and the presence of this modification is largely dependent on producer cell and parameters of manufacturing process. Here we examined and characterized cell culture conditions that determine during cultivation selective changes in galactose content of a model therapeutic mAb IgG1, trastuzumab biosimilar. We demonstrated that such in cultivation process shift of galactosylation does not affect binding of the mAb to its antigen yet modifies interaction of the mAb with Fcγ receptors and therefore enhances antibody dependent cellular cytotoxicity (ADCC).

PFN2a, a new partner of RARα in the cytoplasm.

Retinoic acid receptors (RARs) are classically considered as nuclear ligand-dependent regulators of transcription. Here we highlighted a novel face of the RARα subtype: RARα is present in low amounts in the cytoplasm of mouse embryonic fibroblasts (MEFs) where it interacts with profilin2a (PFN2A), a small actin-binding protein involved in filaments polymerization. The interaction involves the N-terminal proline-rich motif (PRM) of RARα and the SH3-like domain of PFN2a. When increased in the cytoplasm, RARα competes with other PFN2a-binding proteins bearing PRMs and involved in actin filaments elongation. Consequently, the actin filament network is altered and MEFs adhesion is decreased. This novel role opens novel avenues for the understanding of pathologies characterized by increased levels of cytoplasmic RARα.

Nuclear and extranuclear effects of vitamin A.

Vitamin A or retinol is a multifunctional vitamin that is essential at all stages of life from embryogenesis to adulthood. Up to now, it has been accepted that the effects of vitamin A are exerted by active metabolites, the major ones being 11-cis retinal for vision, and all trans-retinoic acid (RA) for cell growth and differentiation. Basically RA binds nuclear receptors, RARs, which regulate the expression of a battery of target genes in a ligand dependent manner. During the last decade, new scenarios have been discovered, providing a rationale for the understanding of other long-noted but not explained functions of retinol. These novel scenarios involve: (i) other nuclear receptors such as PPAR ß/δ, which regulate the expression of other target genes with other functions; (ii) extranuclear and nontranscriptional effects, such as the activation of kinases, which phosphorylate RARs and other transcription factors, thus expanding the list of the RA-activated genes; (iii) finally, vitamin A is active per se and can work as a cytokine that regulates gene transcription by activating STRA6. New effects of vitamin A and RA are continuously being discovered in new fields, revealing new targets and new mechanisms thus improving the understanding the pleiotropicity of their effects.

Nuclear and extra-nuclear effects of retinoid acid receptors: how they are interconnected.

The nuclear retinoic acid receptors (RAR α, ß and γ) and their isoforms are ligand-dependent regulators of transcription Transcription , which mediate the effects of all-trans retinoic acid (RA), the active endogenous metabolite of Vitamin A. They heterodimerize with Retinoid X Receptors (RXRs α, ß and γ), and regulate the expression of a battery of target genes Target genes involved in cell growth and differentiation Differentiation . During the two last decades, the description of the crystallographic structures of RARs, the characterization of the polymorphic response elements of their target genes Target genes , and the identification of the multiprotein complexes involved in their transcriptional activity have provided a wealth of information on their pleiotropic effects. However, the regulatory scenario became even more complicated once it was discovered that RARs are phosphoproteins and that RA can activate kinase signaling cascades via a pool of RARs present in membrane lipid rafts. Now it is known that these RA-activated kinases Kinases translocate to the nucleus where they phosphorylate RARs and other retinoid signaling factors. The phosphorylation Phosphorylation state of the RARs dictates whether the transcriptional programs which are known to be induced by RA are facilitated and/or switched on. Thus, kinase signaling pathways appear to be crucial for fine-tuning the appropriate physiological activity of RARs.

Phosphorylation of the retinoic acid receptor RARγ2 is crucial for the neuronal differentiation of mouse embryonic stem cells.

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest by activating nuclear RA receptors (RARs) (α, ß and γ), which are ligand-dependent transcription factors. RARs are also phosphorylated in response to RA. Here, we investigated the in vivo relevance of the phosphorylation of RARs during RA-induced neuronal differentiation of mouse embryonic stem cells (mESCs). Using ESCs where the genes encoding each RAR subtype had been inactivated, and stable rescue lines expressing RARs mutated in phospho-acceptor sites, we show that RA-induced neuronal differentiation involves RARγ2 and requires RARγ2 phosphorylation. By gene expression profiling, we found that the phosphorylated form of RARγ2 regulates a small subset of genes through binding an unusual RA response element consisting of two direct repeats with a seven-base-pair spacer. These new findings suggest an important role for RARγ phosphorylation during cell differentiation and pave the way for further investigations during embryonic development.

Genes involved in cell adhesion and signaling: a new repertoire of retinoic acid receptor target genes in mouse embryonic fibroblasts.

Nuclear retinoic acid (RA) receptors (RARα, ß and γ) are ligand-dependent transcription factors that regulate the expression of a battery of genes involved in cell differentiation and proliferation. They are also phosphoproteins and we previously showed the importance of their phosphorylation in their transcriptional activity. In the study reported here, we conducted a genome-wide analysis of the genes that are regulated by RARs in mouse embryonic fibroblasts (MEFs) by comparing wild-type MEFs to MEFs lacking the three RARs. We found that in the absence of RA, RARs control the expression of several gene transcripts associated with cell adhesion. Consequently the knockout MEFs are unable to adhere and to spread on substrates and they display a disrupted network of actin filaments, compared with the WT cells. In contrast, in the presence of the ligand, RARs control the expression of other genes involved in signaling and in RA metabolism. Taking advantage of rescue cell lines expressing the RARα or RARγ subtypes (either wild-type or mutated at the N-terminal phosphorylation sites) in the null background, we found that the expression of RA-target genes can be controlled either by a specific single RAR or by a combination of RAR isotypes, depending on the gene. We also selected genes that require the phosphorylation of the receptors for their regulation by RA. Our results increase the repertoire of genes that are regulated by RARs and highlight the complexity and diversity of the transcriptional programs regulated by RARs, depending on the gene.

Vitamin A and retinoid signaling: genomic and nongenomic effects.

Vitamin A or retinol is arguably the most multifunctional vitamin in the human body, as it is essential from embryogenesis to adulthood. The pleiotropic effects of vitamin A are exerted mainly by one active metabolite, all-trans retinoic acid (atRA), which regulates the expression of a battery of target genes through several families of nuclear receptors (RARs, RXRs, and PPARß/δ), polymorphic retinoic acid (RA) response elements, and multiple coregulators. It also involves extranuclear and nontranscriptional effects, such as the activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of several actors of RA signaling. However, vitamin A itself proved recently to be active and RARs to be present in the cytosol to regulate translation and cell plasticity. These new concepts expand the scope of the biologic functions of vitamin A and RA.

Cullin 3 mediates SRC-3 ubiquitination and degradation to control the retinoic acid response.

SRC-3 is an important coactivator of nuclear receptors including the retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated by changes in the posttranslational code of the protein that involves mainly phosphorylation and ubiquitination. We recently reported that SRC-3 is degraded by the proteasome in response to RA. Here, by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL-3 and RBX1 as components of the E3 ubiquitin ligase involved in the RA-induced ubiquitination and subsequent degradation of SRC-3. We also show that the RA-induced ubiquitination of SRC-3 depends on its prior phosphorylation at serine 860 that promotes binding of the CUL-3-based E3 ligase in the nucleus. Finally, phosphorylation, ubiquitination, and degradation of SRC-3 cooperate to control the dynamics of transcription. In all, this process participates to the antiproliferative effect of RA.