Phosphatidylcholine as a metabolic cue for determining B cell fate and function.

In activated B cells, increased production of phosphatidylcholine (PtdCho), the most abundant cellular phospholipid, is handled primarily by the CDP-choline pathway. B cell-specific deletion of CTP:phosphocholine cytidylyltransferase α (CCTα), the rate-limiting enzyme in the CDP-choline pathway, led to augmented IgM secretion and reduced IgG production, suggesting that PtdCho synthesis is required for germinal center reactions. To specifically assess whether PtdCho influences B cell fate during germinal center responses, we examined immune responses in mice whereby PtdCho synthesis is disrupted in B cells that have undergone class switch recombination to IgG1 (referred to as either Cγ1wt/wt, Cγ1Cre/wt or Cγ1Cre/Cre based on Cre copy number). Serum IgG1 was markedly reduced in naïve Cγ1Cre/wt and Cγ1Cre/Cre mice, while levels of IgM and other IgG subclasses were similar between Cγ1Cre/wt and Cγ1wt/wt control mice. Serum IgG2b titers were notably reduced and IgG3 titers were increased in Cγ1Cre/Cre mice compared with controls. Following immunization with T cell-dependent antigen NP-KLH, control mice generated high titer IgG anti-NP while IgG anti-NP titers were markedly reduced in both immunized Cγ1Cre/wt and Cγ1Cre/Cre mice. Correspondingly, the frequency of NP-specific IgG antibody-secreting cells was also reduced in spleens and bone marrow of Cγ1Cre/wt and Cγ. 1Cre/Cre mice compared to control mice. Interestingly, though antigen-specific IgM B cells were comparable between Cγ1Cre/wt, Cγ1Cre/Cre and control mice, the frequency and number of IgG1 NP-specific B cells was reduced only in Cγ1Cre/Cre mice. These data indicate that PtdCho is required for the generation of both germinal center-derived B cells and antibody-secreting cells. Further, the reduction in class-switched ASC but not B cells in Cγ1Cre/wt mice suggests that ASC have a greater demand for PtdCho compared to germinal center B cells.

Regulatory crosstalk within the mammalian unfolded protein response.

Increased demands on the protein folding capacity of the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR). Comprised of a tripartite signaling system, the UPR regulates translation and gene transcription to manifest pro-adaptive and, if necessary, pro-apoptotic outcomes. The three UPR pathways, initiated by activating transcription factor 6, inositol requiring enzyme 1, and protein kinase RNA-activated-like ER kinase (PERK), direct distinct downstream signaling events. However, it is becoming increasingly clear that interplay between the cascades is vital in shaping the UPR. In particular, recent discoveries have revealed that PERK-dependent signals mediate both inter- and intra-pathway regulation within the UPR, underscoring the critical role of the PERK pathway in the cellular response to ER stress.

Micro(RNA)managing endoplasmic reticulum stress.

Cellular disturbances that cause accumulation of misfolded proteins in the endoplasmic reticulum (ER) lead to a condition referred to as "ER stress" and trigger the unfolded protein response (UPR), a signaling pathway that attempts to restore ER homeostasis. The complexity of UPR signaling can generate adaptive and apoptotic outputs, depending on the nature and duration of the ER stress. MicroRNAs (miRNAs), small non-coding RNAs that typically repress gene expression, have recently emerged as key gene regulators of the proadaptive/proapoptotic molecular switch emanating from the ER. Importantly, select miRNAs have been shown to directly regulate key UPR components.

Phospholipids: "greasing the wheels" of humoral immunity.

Phospholipids are major structural components of all cellular membranes. In addition, certain phospholipids execute regulatory activities that affect cell behavior, function and fate in critically important physiological settings. The influence of phospholipids is especially obvious in the adaptive immune system, where these macromolecules mediate both intrinsic and extrinsic effects on B and T lymphocytes. This review article highlights the action of lysophospholipid sphingosine-1-phosphate as a lymphocyte chemoattractant, the function of phosphatidylinositol phosphates as signaling conduits in lymphocytes and the role of phospholipids as raw materials for membrane assembly and organelle biogenesis in activated B lymphocytes. Special emphasis is placed on the means by which these three processes push humoral immune responses forward. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

The specialized unfolded protein response of B lymphocytes: ATF6α-independent development of antibody-secreting B cells.

B lymphocytes, like all mammalian cells, are equipped with the unfolded protein response (UPR), a complex signaling system allowing for both pro- and mal-adaptive responses to increased demands on the endoplasmic reticulum (ER). The UPR is comprised of three signaling pathways initiated by the ER transmembrane stress sensors, IRE1α/ß, PERK and ATF6α/ß. Activation of IRE1 yields XBP1(S), a transcription factor that directs expansion of the ER and enhances protein biosynthetic and secretory machinery. XBP1(S) is essential for the differentiation of B lymphocytes into antibody-secreting cells. In contrast, the PERK pathway, a regulator of translation and transcription, is dispensable for the generation of antibody-secreting cells. Functioning as a transcription factor, ATF6α can augment ER quality control processes and drive ER expansion, but the potential role of this UPR pathway in activated B cells has not been investigated. Here, we report studies of ATF6α-deficient B cells demonstrating that ATF6α is not required for the development of antibody-secreting cells. Thus, when B cells are stimulated to secrete antibody, a specialized UPR relies exclusively on the IRE1-XBP1 pathway to remodel the ER and expand cellular secretory capacity.

MicroRNA-30c-2* limits expression of proadaptive factor XBP1 in the unfolded protein response.

Stress in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a multifaceted signaling system coordinating translational control and gene transcription to promote cellular adaptation and survival. Microribonucleic acids (RNAs; miRNAs), single-stranded RNAs that typically function as posttranscriptional modulators of gene activity, have been shown to inhibit translation of certain secretory pathway proteins during the UPR. However, it remains unclear whether miRNAs regulate UPR signaling effectors directly. In this paper, we report that a star strand miRNA, miR-30c-2* (recently designated miR-30c-2-3p), is induced by the protein kinase RNA activated-like ER kinase (PERK) pathway of the UPR and governs expression of XBP1 (X-box binding protein 1), a key transcription factor that augments secretory capacity and promotes cell survival in the adaptive UPR. These data provide the first link between an miRNA and direct regulation of the ER stress response and reveal a novel molecular mechanism by which the PERK pathway, via miR-30c-2*, influences the scale of XBP1-mediated gene expression and cell fate in the UPR.

UPR-Mediated Membrane Biogenesis in B Cells.

The unfolded protein response (UPR) can coordinate the regulation of gene transcription and protein translation to balance the load of client proteins with the protein folding and degradative capacities of the ER. Increasing evidence also implicates the UPR in the regulation of lipid synthesis and membrane biogenesis. The differentiation of B lymphocytes into antibody-secreting cells is marked by significant expansion of the ER, the site for antibody synthesis and assembly. In activated B cells, the demand for membrane protein and lipid components leads to activation of the UPR transcriptional activator XBP1(S) which, in turn, initiates a cascade of biochemical events that enhance supplies of phospholipid precursors and build machinery for the synthesis, maturation, and transport of secretory proteins. The alterations in lipid metabolism that occur during this developmental transition and the impact of membrane phospholipid restriction on B cell secretory characteristics are discussed in this paper.

Intricately Regulated: A Cellular Toolbox for Fine-Tuning XBP1 Expression and Activity.

Stress in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a signaling mechanism that allows cellular adaptation to ER stress by engaging pro-adaptive transcription factors and alleviating protein folding demand. One such transcription factor, X-box binding protein (XBP1), originates from the inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1) UPR stress sensor. XBP1 up-regulates a pool of genes involved in ER protein translocation, protein folding, vesicular trafficking and ER- associated protein degradation. Recent data suggest that the regulation of XBP1 expression and transcriptional activity may be a tissue- and stress-dependent phenomenon. Moreover, the intricacies involved in “fine-tuning” XBP1 activity in various settings are now coming to light. Here, we provide an overview of recent developments in understanding the regulatory mechanisms underlying XBP1 expression and activity and discuss the significance of these new insights.

The unfolded protein response (UPR)-activated transcription factor X-box-binding protein 1 (XBP1) induces microRNA-346 expression that targets the human antigen peptide transporter 1 (TAP1) mRNA and governs immune regulatory genes.

To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3'-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.

ATF6alpha induces XBP1-independent expansion of the endoplasmic reticulum.

A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6alpha (ATF6alpha), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6alpha drives ER expansion and can do so in the absence of XBP1(S). Overexpression of active ATF6alpha induces PtdCho biosynthesis and modulates the CDP-choline pathway differently than does enforced expression of XBP1(S). These data indicate that ATF6alpha and XBP1(S) have the ability to regulate lipid biosynthesis and ER expansion by mechanisms that are at least partially distinct. These studies reveal further complexity in the potential relationships between UPR pathways, lipid production and ER biogenesis.

CTP:phosphocholine cytidylyltransferase alpha is required for B-cell proliferation and class switch recombination.

CTP:phosphocholine cytidylyltransferase (CCT) is a key rate-controlling enzyme in the biosynthetic pathway leading to the principle membrane phospholipid, phosphatidylcholine. CCTalpha is the predominant isoform expressed in mammalian cells. To investigate the role of CCTalpha in the development and function of B-lymphocytes, mice with B-lymphocytes that selectively lacked CCTalpha were derived using the CD19-driven Cre/loxP system. When challenged with a T-cell-dependent antigen, the animals harboring CCTalpha-deficient B-cells exhibited a hyper-IgM secretion phenotype coupled with a lack of IgG production. The inability of CCTalpha-/- B-cells to undergo class switch recombination correlated with a proliferation defect in vivo and in vitro in response to antigenic and mitogenic stimuli. Lipopolysaccharide stimulation of CCTalpha-/- B-cells resulted in an early trigger of the unfolded protein response-mediated splicing of Xbp-1 mRNA, and this was accompanied by accelerated kinetics of IgM secretion and higher incidence of IgM-secreting cells. Thus, the inability of stimulated B-cells to produce enough phosphatidylcholine prevents proliferation and class switch recombination but leads to unfolded protein response activation and a hyper-IgM secretion phenotype.

Coronavirus infection modulates the unfolded protein response and mediates sustained translational repression.

During coronavirus replication, viral proteins induce the formation of endoplasmic reticulum (ER)-derived double-membrane vesicles for RNA synthesis, and viral structural proteins assemble virions at the ER-Golgi intermediate compartment. We hypothesized that the association and intense utilization of the ER during viral replication would induce the cellular unfolded protein response (UPR), a signal transduction cascade that acts to modulate translation, membrane biosynthesis, and the levels of ER chaperones. Here, we report that infection by the murine coronavirus mouse hepatitis virus (MHV) triggers the proximal UPR transducers, as revealed by monitoring the IRE1-mediated splicing of XBP-1 mRNA and the cleavage of ATF6alpha. However, we detected minimal downstream induction of UPR target genes, including ERdj4, ER degradation-enhancing alpha-mannosidase-like protein, and p58(IPK), or expression of UPR reporter constructs. Translation initiation factor eIF2alpha is highly phosphorylated during MHV infection, and translation of cellular mRNAs is attenuated. Furthermore, we found that the critical homeostasis regulator GADD34, which recruits protein phosphatase 1 to dephosphorylate eIF2alpha during the recovery phase of the UPR, is not expressed during MHV infection. These results suggest that MHV modifies the UPR by impeding the induction of UPR-responsive genes, thereby favoring a sustained shutdown of the synthesis of host cell proteins while the translation of viral proteins escalates. The role of this modified response and its potential relevance to viral mechanisms for the evasion of innate defense signaling pathways during coronavirus replication are discussed.

The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells.

When B-lymphocytes differentiate into plasma cells, immunoglobulin (Ig) heavy and light chain synthesis escalates and the entire secretory apparatus expands to support high-rate antibody secretion. These same events occur when murine B-cells are stimulated with lipopolysaccharide (LPS), providing an in vitro model in which to investigate the differentiation process. The unfolded protein response (UPR), a multi-pathway signaling response emanating from the endoplasmic reticulum (ER) membrane, allows cells to adapt to increasing demands on the protein folding capacity of the ER. As such, the UPR plays a pivotal role in the differentiation of antibody-secreting cells. Three specific stress sensors, IRE1, PERK/PEK and ATF6, are central to the recognition of ER stress and induction of the UPR. IRE1 triggers splicing of Xbp-1 mRNA, yielding a transcriptional activator of the UPR termed XBP-1(S), and activation of the IRE1/XBP-1 pathway has been reported to be required for expansion of the ER and antibody secretion. Here, we provide evidence that PERK is not activated in LPS-stimulated splenic B-cells, whereas XBP-1(S) and the UPR transcriptional activator ATF6 are both induced. We further demonstrate that Perk-/- B-cells develop and are fully competent for induction of Ig synthesis and antibody secretion when stimulated with LPS. These data provide clear evidence for differential activation and utilization of distinct UPR components as activated B-lymphocytes increase Ig synthesis and differentiate into specialized secretory cells.

Phospholipid biosynthesis program underlying membrane expansion during B-lymphocyte differentiation.

Stimulated B-lymphocytes differentiate into plasma cells committed to antibody production. Expansion of the endoplasmic reticulum and Golgi compartments is a prerequisite for high rate synthesis, assembly, and secretion of immunoglobulins. The bacterial cell wall component lipopolysaccharide (LPS) stimulates murine B-cells to proliferate and differentiate into antibody-secreting cells that morphologically resemble plasma cells. LPS activation of CH12 B-cells augmented phospholipid production and initiated a genetic program, including elevated expression of the genes for the synthesis, elongation, and desaturation of fatty acids that supply the phospholipid acyl moieties. Likewise, many of the genes in phospholipid biosynthesis were up-regulated, most notably those encoding Lipin1 and choline phosphotransferase. In contrast, CTP:phosphocholine cytidylyltransferase alpha (CCTalpha) protein, a key control point in phosphatidylcholine biosynthesis, increased because of stabilization of protein turnover rather than transcriptional activation. Furthermore, an elevation in cellular diacylglycerol and fatty acid correlated with enhanced allosteric activation of CCTalpha by the membrane lipids. This work defines a genetic and biochemical program for membrane phospholipid biogenesis that correlates with an increase in the phospholipid components of the endoplasmic reticulum and Golgi compartments in LPS-stimulated B-cells.

Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis.

Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein response-mediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDP-choline pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis.

Evidence that marginal zone B cells possess an enhanced secretory apparatus and exhibit superior secretory activity.

Marginal zone B (MZB) cells are the first splenic B cells to initiate Ab secretion against polysaccharide-encapsulated Ags in vivo. This swift MZB cell response can be reproduced in vitro as LPS treatment induces Ab secretion in as little as 12 h. Conversely, in vitro LPS treatment of splenic follicular B (FOB) cells results in Ab secretion after 2-3 days. The basis for these distinct response kinetics is not understood. We performed ex vivo analysis of resting and LPS-stimulated murine MZB and FOB cells and found that MZB cells express higher levels of the LPS TLR complex RP105/MD-1 and respond to much lower concentrations of LPS than do FOB cells. Furthermore, increasing doses of LPS do not accelerate the kinetics by which FOB cells transition into Ab secretion. Ultrastructural analysis of resting cells demonstrated that rough endoplasmic reticulum is more abundant in MZB cells than in FOB cells. Additionally, RT-PCR and immunoblot analyses revealed that numerous endoplasmic reticulum resident chaperones and folding enzymes are expressed at greater levels in resting MZB cells than in resting FOB cells. Although both LPS-stimulated MZB and FOB cells increase expression of these factors, MZB cells exhibit a more rapid increase that correlates with accelerated kinetics of Ab secretion and higher per cell output of secreted IgM. These data indicate that MZB cells are equipped for exquisite sensitivity to bacterial components like LPS and poised for rapid, robust Ab production, making MZB cells ideally suited as frontline defenders in humoral immunity.

Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation.

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.

Building an antibody factory: a job for the unfolded protein response.

Plasma cells are highly specialized, terminally differentiated secretory cells that produce tremendous quantities of a single product, the antibody molecule. In differentiating from a quiescent B cell, the plasma cell must undergo a dramatic architectural metamorphosis. This process entails augmenting the secretory organelles and the proteins that populate them, upregulating their energy and translation potential, and increasing the quality control system to do the job. This transformation is accomplished by an interplay between B lineage-specific transcriptional programs that control plasma cell differentiation and an unfolded protein response.

XBP1: a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum.

When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation. Here, we report that enforced expression of XBP1(S), the active form of the XBP1 transcription factor generated by UPR-mediated splicing of XBP1 mRNA, is sufficient to induce synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis. These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis.