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Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-α

Estrogen deficiency induces bone loss by upregulating osteoclastogenesis by mechanisms not completely defined. We found that ovariectomy-enhanced T-cell production of TNF-alpha, which, acting through the TNF-alpha receptor p55, augments macrophage colony-stimulating factor-induced (M-CSF-induced) and RANKL-induced osteoclastogenesis. Ovariectomy failed to induce bone loss, stimulate bone resorption, or increase M-CSF- and RANKL-dependent osteoclastogenesis in T-cell deficient mice, establishing T cells as essential mediators of the bone-wasting effects of estrogen deficiency in vivo. These findings demonstrate that the ability of estrogen to target T cells, suppressing their production of TNF-alpha, is a key mechanism by which estrogen prevents osteoclastic bone resorption and bone loss.

Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo.

In vivo studies have shown T cells to be central to the mechanism by which estrogen deficiency induces bone loss, but the mechanism involved remains, in part, undefined. In vitro, T cells from ovariectomized mice produce increased amounts of tumor necrosis factor (TNF), which augments receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. However, both the mechanism and the relevance of this phenomenon in vivo remain to be established. In this study, we found that ovariectomy increased the number of bone marrow T cell-producing TNF without altering production of TNF per T cell. Attesting to the essential contribution of TNF, ovariectomy induced rapid bone loss in wild type (wt) mice but failed to do so in TNF-deficient (TNF−/−) mice. Furthermore, ovariectomy induced bone loss, which was absent in T cell-deficient nude mice, was restored by adoptive transfer of wt T cells, but not by reconstitution with T cells from TNF−/− mice. These findings demonstrate the key causal role of T cell-produced TNF in the bone loss after estrogen withdrawal. Finally, ovariectomy caused bone loss in wt mice and in mice lacking p75 TNF receptor but failed to do so in mice lacking the p55 TNF receptor. These findings demonstrate that enhanced T cell production of TNF resulting from increased bone marrow T cell number is a key mechanism by which estrogen deficiency induces bone loss in vivo. The data also demonstrate that the bone-wasting effect of TNF in vivo is mediated by the p55 TNF receptor.

Estrogen decreases osteoclast formation by down-regulating receptor activator of NF-kappa B ligand (RANKL)-induced JNK activation.

The differentiation of cells of the monocytic lineage into mature osteoclasts (OC) is specifically induced by the tumor necrosis factor-related factor, RANKL (receptor activator of NF-κB ligand; also known as OPGL, ODF, or TRANCE). Because inhibition of osteoclastogenesis is one of the main mechanisms by which estrogen (E2) prevents bone loss, it is likely that E2 may regulate either the production of, or the target cell responsiveness to RANKL. We found that E2 decreases the differentiation into OC of both murine bone marrow monocytes and RAW 264.7 cells, a monocytic line, by down-regulating the activation of Jun N-terminal kinase 1 (JNK1). Diminished JNK1 activity results in decreased nuclear levels of the key osteoclastogenic transcription factors, c-Fos and c-Jun, and lower binding of these transcriptional inducers to DNA. Thus, one novel mechanism by which E2 down-regulates osteoclastogenesis is by decreasing the responsiveness of OC precursors to RANKL.

Estrogen decreases TNF gene expression by blocking JNK activity and the resulting production of c-Jun and JunD

Central to the bone-sparing effect of estrogen (E(2)) is its ability to block the monocytic production of the osteoclastogenic cytokine TNF-alpha (TNF). However, the mechanism by which E(2) downregulates TNF production is presently unknown. Transient transfection studies in HeLa cells, an E(2) receptor-negative line, suggest that E(2) inhibits TNF gene expression through an effect mediated by estrogen receptor beta (ERbeta). We also report that in RAW 264.7 cells, an E(2) receptor-positive murine monocytic line, E(2) downregulates cytokine-induced TNF gene expression by decreasing the activity of the Jun NH(2)-terminal kinase (JNK). The resulting diminished phosphorylation of c-Jun and JunD at their NH(2)-termini decreases the ability of these nuclear proteins to autostimulate the expression of the c-Jun and JunD genes, thus leading to lower production of c-Jun and JunD. The consequent decrease in the nuclear levels of c-Jun and JunD leads to diminished binding of c-Jun/c-Fos and JunD/c-Fos heterodimers to the AP-1 consensus sequence in the TNF promoter and, thus, to decreased transactivation of the TNF gene.

Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-γ-induced class II transactivator

Expansion of the pool of tumor necrosis factor (TNF)-α-producing T cells is instrumental for the bone loss induced by estrogen deficiency, but the responsible mechanism is unknown. Here we show that ovariectomy up-regulates IFN-γ-induced class II transactivator, a multitarget immune modulator, resulting in increased antigen presentation by macrophages, enhanced T cell activation, and prolonged lifespan of active T cells. Up-regulation of class II transactivator derives from increased production of IFN-γ by T helper 1 cells, resulting from enhanced secretion of IL-12 and IL-18 by macrophages. The resulting T cell expansion and bone loss are prevented in vivo by both blockade of antigen presenting cell-induced T cell activation, and silencing of IFN-γ receptor signaling. Thus, increased IFN-γ-induced class II transactivator expression and the resulting enhanced T cell proliferation and lifespan are critical to the bone wasting effect of estrogen deficiency.

Plasma cells require autophagy for sustainable immunoglobulin production

The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5−/− differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.

The proteasome load versus capacity balance determines apoptotic sensitivity of multiple myeloma cells to proteasome inhibition

Proteasome inhibitors (PIs) are effective against multiple myeloma (MM), but the mechanisms of action and bases of individual susceptibility remain unclear. Recent work linked PI sensitivity to protein synthesis and proteasome activity, raising the question whether different levels of proteasome expression and workload underlie PI sensitivity in MM cells (MMCs). Exploiting human MM lines characterized by differential PI sensitivity, we report that highly sensitive MMCs express lower proteasome levels and higher proteasomal workload than relatively PI-resistant MMCs, resulting in the accumulation of polyubiquitinated proteins at the expense of free ubiquitin (proteasome stress). Manipulating proteasome expression or workload alters apoptotic sensitivity to PI, demonstrating a cause-effect relationship between proteasome stress and apoptotic responses in MMCs. Intracellular immunostaining in primary, patient-derived MMCs reveals that polyubiquitinated proteins hallmark neoplastic plasma cells, in positive correlation with immunoglobulin (Ig) content, both intra- and interpatient. Moreover, overall proteasome activity of primary MMCs inversely correlates with apoptotic sensitivity to PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMCs to PI, potentially providing a framework for identifying indicators of responsiveness and designing novel combination therapies.

T cell activation induces human osteoclast formation via receptor activator of nuclear factor κB ligand-dependent and -independent mechanisms

In unstimulated conditions, osteoclast (OC) formation is regulated by stromal cell production of the key osteoclastogenic factors receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). However, the mechanisms of accelerated osteoclastogenesis and bone loss characteristic of inflammatory conditions are poorly understood but appear to involve T cells. In addition, the mechanism by which OCs arise spontaneously in cultures of peripheral blood mononuclear cells in the absence of stromal cells or added cytokines remains unclear. Using a stromal cell free human osteoclast generating system, we investigated the ability of activated T cells to support osteoclastogenesis. We show that when activated by phytohemagglutinin‐P (PHA), T cells (both CD4+ and CD8+) stimulate human OC formation in vitro. Although both soluble M‐CSF and RANKL were detected in activated T cell supernatants, the presence of M‐CSF was not essential for macrophage survival or RANKL‐dependent osteoclast formation, suggesting that other soluble T cell‐derived factors were capable of substituting for this cytokine. We also found that saturating concentrations of osteoprotegerin (OPG) failed to neutralize 30% of the observed OC formation and that T cell conditioned medium (CM) could superinduce osteoclastogenesis in cultures of purified monocytes maximally stimulated by RANKL and M‐CSF. Together, these data suggest that activated T cells support osteoclastogenesis via RANKL‐dependent and ‐independent mechanisms. Although not relevant for T cell‐induced osteoclastogenesis, secretion of soluble M‐CSF is a previously undescribed property of activated T cells.

Progressively impaired proteasomal capacity during terminal plasma cell differentiation

After few days of intense immunoglobulin (Ig) secretion, most plasma cells undergo apoptosis, thus ending the humoral immune response. We asked whether intrinsic factors link plasma cell lifespan to Ig secretion. Here we show that in the late phases of plasmacytic differentiation, when antibody production becomes maximal, proteasomal activity decreases. The excessive load for the reduced proteolytic capacity correlates with accumulation of polyubiquitinated proteins, stabilization of endogenous proteasomal substrates (including Xbp1s, IκBα, and Bax), onset of apoptosis, and sensitization to proteasome inhibitors (PI). These events can be reproduced by expressing Ig‐μ chain in nonlymphoid cells. Our results suggest that a developmental program links plasma cell death to protein production, and help explaining the peculiar sensitivity of normal and malignant plasma cells to PI.

Managing and exploiting stress in the antibody factory

Like us, our cells have evolved strategies to cope with, and sometimes utilize, stress. Molecular analyses of plasma cell biogenesis, lifestyle and death suggest that protein synthesis‐dependent stress is utilised to integrate differentiation, function and lifespan control. Plasma cells are short‐lived professional secretory cells, each of them capable of releasing several thousands antibodies per second. Their differentiation from B lymphocytes entails the spectacular enlargement of the endoplasmic reticulum (ER), finalized to sustain massive Ig production. Nonetheless, symptoms of ER stress are evident, and the UPR‐related transcription factor XBP‐1 is essential for differentiation. Surprisingly, the development of such an efficient factory is matched by a decrease in proteasomes. The unbalanced load/capacity ratio leads to accumulation of polyubiquitinated molecules and predisposes plasma cells to apoptosis. Exuberant antibody secretion imposes considerable stress on metabolic and redox homeostasis. Collectively, these stressful conditions may link plasma cell death to antibody production, providing a molecular counter for secreted molecules, as well as an explanation for the peculiar sensitivity of myeloma cells towards proteasome inhibitors.