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Joseph W Brewer

Publications and source records attributed to Joseph W Brewer.

11 recordsLinked to original sources
PubMed2007

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.

Animals↗
PubMed2007

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.

Animals↗
PubMed2006

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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2004

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.

3T3 Cells↗
PubMed2004

Stressed-out B cells? Plasma-cell differentiation and the unfolded protein response.

Plasma cells operate as factories where large quantities of Ig heavy and light chains are made and assembled into functional antibodies. The finished products are shipped out with impressive efficiency. A major component of the machinery necessary for high-rate antibody secretion is an elaborate network of endoplasmic reticulum (ER), the site of antibody biosynthesis. Recent discoveries have provided insights into how this expansive secretory machinery is built, equipped and maintained. The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the ER, regulates the expression and activity of X-box binding protein 1, a transcription factor required for plasma-cell development. The UPR pathway therefore senses conditions in the ER--the very compartment where antibodies are formed--and directs events required for humoral immunity.

Animals↗
PubMed2004

A role for the unfolded protein response in optimizing antibody secretion.

Terminal differentiation of B lymphocytes into antibody(Ab)-secreting plasma cells is marked by a sharp rise in immunoglobulin (Ig) biosynthesis that increases demand on the protein folding capacity of the endoplasmic reticulum (ER). The unfolded protein response pathway (UPR) allows cells to respond to challenging conditions within the ER, in part by the activities of the XBP1 and ATF6alpha/beta transcription factors. The UPR is activated in differentiating B cells, and XBP1 is required for the generation of Ab-secreting plasma cells. Therefore, it has been hypothesized that the UPR mediates ER homeostasis as B cells transition into high-rate Ab secretion. We sought to test this hypothesis in primary murine splenic B cells stimulated to secrete Ab in vitro. Here, we report that enforced expression of a dominant-negative ATF6alpha mutant in differentiating B cells reduces the output of secreted IgM and increases improper release of IgM assembly intermediates. These data indicate that the UPR functions to optimize the efficiency of Ab secretion and provide new insight into the fundamental role of the UPR in humoral immunity.

Activating Transcription Factor 6↗
PubMed2002

Activation of an unfolded protein response during differentiation of antibody-secreting B cells.

The unfolded protein response pathway (UPR) is believed to detect and compensate for excessive protein accumulation in the endoplasmic reticulum (ER). The UPR can be induced by pharmacological agents that perturb ER functions, but may also occur during cellular developmental processes such as the transition of B-lymphocytes into antibody-secreting plasma cells. Here we show that major UPR components are activated in B cells stimulated to secrete antibody. Increased expression of UPR targets including the ER chaperones BiP and GRP94 and the transcription factor XBP-1 initiates early in the differentiation program prior to up-regulated synthesis of Ig chains. Furthermore, these same kinetics are observed during differentiation for cleavage of the ER-localized ATF6alpha protein and splicing of XBP-1 mRNA to generate p50ATF6alpha and p54XBP-1, the two known UPR transcriptional activators. All of these UPR events reach maximal levels once Ig synthesis and secretion are markedly induced. Interestingly, these events are not accompanied by expression of CHOP, a transcription factor induced by ER stress agents commonly used to investigate the UPR. These results suggest that a physiological UPR elicited during differentiation of B-lymphocytes into high-rate secretory cells may be distinct from the UPR defined by agents that disrupt protein maturation in the ER.

Antibodies↗
PubMed2002

Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response.

CHOP is a non-ER localized transcription factor that is induced by a variety of adverse physiological conditions including ER stress. Accumulation of unfolded proteins in the ER activates an unfolded protein response pathway that targets both ER resident chaperones (e.g. BiP) and CHOP. Hence, it is unclear if CHOP induction during ER stress occurs through the ER stress response element that is conserved in both CHOP and ER chaperone promoters, or through a separate regulatory pathway conserved among different CHOP inducing cellular stress conditions. We identified a bona fide ER stress element in the hamster CHOP promoter and found that similar transcription complexes containing NF-Y bound to both the CHOP and BiP ER stress response elements. In addition, we demonstrated for the first time the importance of the C/EBP-ATF composite site for CHOP regulation during ER stress. Activation of the ER transmembrane eIF2alpha kinase, PERK, induced ATF4 protein expression, direct binding to the composite site in CHOP promoter, and as a consequence, CHOP protein induction. We propose that this eIF2alpha-kinase/ATF4/C/EBP-ATF composite site pathway is conserved for CHOP regulation during various cellular stress conditions including ER stress. Our data indicate that both the ERSE and the PERK-ATF4 pathways converge on the CHOP promoter during ER stress and provide insights into the similarities and differences between CHOP and ER chaperone expression during normal and stress conditions.

Animals↗