Role of c-Fos in orthodontic tooth movement: an in vivo study using transgenic mice.

OBJECTIVES: The transcription factor c-Fos controls the differentiation of osteoclasts and is expressed in periodontal ligament cells after mechanical stimulation in vitro. However, it is unclear how c-Fos regulates orthodontic tooth movement (OTM) in vivo. The aim of this study was therefore to analyse OTM in transgenic mice with overexpression of c-Fos. MATERIALS AND METHODS: We employed c-Fos transgenic mice (c-Fos tg) and wild-type littermates (WT) in a model of OTM induced by Nitinol tension springs that were bonded between the left first maxillary molars and the upper incisors. The unstimulated contralateral side served as an internal control. Mice were analysed by contact radiography, micro-computed tomography, decalcified histology and histochemistry. RESULTS: Our analysis of the unstimulated side revealed that alveolar bone and root morphology were similar between c-Fos tg and control mice. However, we observed more osteoclasts in the alveolar bone of c-Fos tg mice as tartrate-resistant acid phosphatase (TRAP)-positive cells were increased by 40%. After 12 days of OTM, c-Fos tg mice exhibited 62% increased tooth movement as compared with WT mice. Despite the faster tooth movement, c-Fos tg and WT mice displayed the same amount of root resorption. Importantly, we did not observe orthodontically induced tissue necrosis (i.e. hyalinization) in c-Fos tg mice, while this was a common finding in WT mice. CONCLUSION: Overexpression of c-Fos accelerates tooth movement without causing more root resorption. CLINICAL RELEVANCE: Accelerated tooth movement must not result in more root resorption as higher tissue turnover may decrease the amount of mechanically induced tissue necrosis.

The high bone mass phenotype of Lrp5-mutant mice is not affected by megakaryocyte depletion.

Bone remodeling is a continuously ongoing process mediated by bone-resorbing osteoclasts and bone-forming osteoblasts. One key regulator of bone formation is the putative Wnt co-receptor Lrp5, where activating mutations in the extracellular domain cause increased bone formation in mice and humans. We have previously reported that megakaryocyte numbers are increased the bone marrow of mice carrying a high bone mass mutation (HBM) of Lrp5 (Lrp5G170V). Since megakaryocytes can promote bone formation, we addressed the question, if the bone remodeling phenotype of Lrp5G170V mice is affected by megakaryocyte depletion. For that purpose we took advantage of a mouse model carrying a mutation of the Mpl gene, encoding the thrombopoietin receptor. These mice (Mplhlb219) were crossed with Lrp5G170V mice to generate animals carrying both mutations in a homozygous state. Using µCT, undecalcified histology and bone-specific histomorphometry of 12 weeks old littermates we observed that megakaryocyte number was remarkably decreased in Mplhlb219/Lrp5G170V mice, yet the high bone mass phenotype of Lrp5G170V mice was not significantly affected by the homozygous Mpl mutation. Finally, when we analyzed 24 weeks old wildtype and Mplhlb219 mice we did not observe a statistically significant alteration of bone remodeling in the latter ones. Taken together, our results demonstrate that an increased number of bone marrow megakaryocytes does not contribute to the increased bone formation caused by Lrp5 activation.

Rsk2 controls synovial fibroblast hyperplasia and the course of arthritis.

OBJECTIVE: Arthritis is a chronic inflammatory disease characterised by immune cell infiltration and mesenchymal cell expansion in the joints. Although the role of immune cells in arthritis is well characterised, the development of mesenchymal cell hyperplasia needs to be better defined. Here, we analysed the role of the ribosomal S6 kinase Rsk2, which we found to be highly activated in joints of patients with arthritis, in the development of mesenchymal cell hyperplasia. METHODS: We genetically inactivated Rsk2 in the tumour necrosis factor (TNF)-α transgenic (TNFtg) mice, an animal model for human inflammatory arthritis. Clinical and histological signs of arthritis as well as molecular markers of inflammation and joint destruction were quantified. Fibroblast-like synoviocytes (FLS) were characterised in vitro and the effect of Rsk2 deletion on the pattern of gene expression was determined. RESULTS: Rsk2 deficiency in TNFtg mice results in earlier and exacerbated inflammation as well as increased bone and cartilage destruction. The production of inflammatory cytokines, matrix metalloproteinases and osteoclastogenic molecules was significantly increased in vivo upon Rsk2 inactivation. Bone marrow deficient in Rsk2 could not transfer this phenotype, indicating that Rsk2 expression in mesenchymal cells controls the course of arthritis. Indeed, Rsk2 deficiency was associated with a more activated phenotype and higher proliferative capacity of FLS, thereby increasing cytokines and production of matrix proteinases. CONCLUSIONS: Rsk2 emerges as a key regulator of mesenchymal cell numbers in the joint and thereby could be targeted to control the inflammatory and tissue-destructive feature of joints in arthritis.

Inhibition of bone remodeling in young mice by bisphosphonate displaces the plasma cell niche into the spleen.

The bone marrow provides niches for early B cell differentiation and long-lived plasma cells. Therefore, it has been hypothesized that perturbing bone homeostasis might impact B cell function and Ab production. This hypothesis is highly relevant for patients receiving long-term treatment with antiresorptive drugs. We therefore analyzed the humoral immune response of mice chronically treated with ibandronate, a commonly used bisphosphonate. We confirmed the increased bone mass caused by inhibition of osteoclast activity and also the strongly reduced bone formation because of decreased osteoblast numbers in response to ibandronate. Thus, bisphosphonate drastically inhibited bone remodeling. When ibandronate was injected into mice after a primary immunization to mimic common antiosteoporotic treatments, the generation of the various B cell populations, the response to booster immunization, and the generation of plasma cells were surprisingly normal. Mice also responded normally to immunization when ibandronate was applied to naive mice. However, there, ibandronate shunted the homing of bone marrow plasma cells. Interestingly, ibandronate reduced the numbers of megakaryocytes, a known component of the bone marrow plasma cell niche. In line with normal Ab responses, increased plasma cell populations associated with increased megakaryocyte numbers were then observed in the spleens of the ibandronate-treated mice. Thus, although inhibition of bone remodeling disturbed the bone marrow plasma cell niche, a compensatory niche may have been created by relocating the megakaryocytes into the spleen, thereby allowing normal B cell responses. Therefore, megakaryocytes may act as a key regulator of plasma cell niche plasticity.

Inflammatory monocytes and Fcγ receptor IV on osteoclasts are critical for bone destruction during inflammatory arthritis in mice.

Destruction of bone tissue by osteoclasts represents a severe pathological phenotype during inflammatory arthritis and results in joint pain and bone malformations. Previous studies have established the essential role of cytokines including TNFα and receptor-ligand interactions, such as the receptor activator of nuclear factor-kappa B-receptor activator of nuclear factor-kappa B ligand interaction for osteoclast formation during joint inflammation. Moreover, autoantibodies contribute to joint inflammation in inflammatory arthritis by triggering cellular fragment crystallizable (Fc)γ receptors (FcγR), resulting in the release of proinflammatory cytokines and chemokines essential for recruitment and activation of innate immune effector cells. In contrast, little is known about the expression pattern and function of different FcγRs during osteoclast differentiation. This would allow osteoclasts to directly interact with autoantibody immune complexes, rather than being influenced indirectly via proinflammatory cytokines released upon immune complex binding to other FcγR-expressing innate immune cells. To address this question, we studied FcγR expression and function on osteoclasts during the steady state and during acute joint inflammation in a model of inflammatory arthritis. Our results suggest that osteoclastogenesis is directly influenced by IgG autoantibody binding to select activating FcγRs on immature osteoclasts, resulting in enhanced osteoclast generation and, ultimately, bone destruction.

Increased skeletal VEGF enhances beta-catenin activity and results in excessively ossified bones.

Vascular endothelial growth factor (VEGF) and beta-catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone-related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over-expression in osteo-chondroprogenitors and their progeny largely pheno-copied constitutive beta-catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2- and phosphatidyl inositol 3-kinase-mediated pathway inducing beta-catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3-beta phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate beta-catenin activity may have widespread physiological and clinical ramifications.

RSK2 protects mice against TNF-induced bone loss.

Tumor necrosis factor (TNF)-α is a key cytokine regulator of bone and mediates inflammatory bone loss. The molecular signaling that regulates bone loss downstream of TNF-α is poorly defined. Here, we demonstrate that inactivating the pro-osteoblastogenic ERK-activated ribosomal S6 kinase RSK2 leads to a drastically accelerated and amplified systemic bone loss in mice ectopically expressing TNF-α [human TNF transgenic (hTNFtg) mice]. The phenotype is associated with a decrease in bone formation because of fewer osteoblasts as well as a drastically increased bone destruction by osteoclasts. The molecular basis of this phenotype is a cell autonomous increased sensitivity of osteoblasts and osteocytes to TNF-induced apoptosis combined with an enhancement of their osteoclast supportive activity. Thus, RSK2 exerts a strong negative regulatory loop on TNF-induced bone loss.

Elevated Fra-1 expression causes severe lipodystrophy.

A shift from osteoblastogenesis to adipogenesis is one of the underlying mechanisms of decreased bone mass and increased fat during aging. We now uncover a new role for the transcription factor Fra-1 in suppressing adipogenesis. Indeed, Fra1 (Fosl1) transgenic (Fra1tg) mice, which developed progressive osteosclerosis as a result of accelerated osteoblast differentiation, also developed a severe general lipodystrophy. The residual fat of these mice appeared immature and expressed lower levels of adipogenic markers, including the fatty acid transporter Cd36 and the CCAAT/enhancer binding protein Cebpa. Consequently accumulation of triglycerides and free fatty acids were detected in the serum of fasting Fra1tg mice. Fra-1 acts cell autonomously because the adipogenic differentiation of Fra1 transgenic primary osteoblasts was drastically reduced, and overexpression of Fra-1 in an adipogenic cell line blocked their differentiation into adipocytes. Strikingly, Cebpa was downregulated in the Fra-1-overexpressing cells and Fra-1 could bind to the Cebpa promoter and directly suppress its activity. Thus, our data add to the known common systemic control of fat and bone mass, a new cell-autonomous level of control of cell fate decision by which the osteogenic transcription factor Fra-1 opposes adipocyte differentiation by inhibiting C/EBPα.

IL-33 shifts the balance from osteoclast to alternatively activated macrophage differentiation and protects from TNF-alpha-mediated bone loss.

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2(-/-) bone marrow led to more bone loss compared with the chimeras with ST2(+/+) bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206(+) AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

Regulatory T cells protect from local and systemic bone destruction in arthritis.

We previously demonstrated the suppressive effects of regulatory T cells (Treg cells) on osteoclast differentiation in vitro. In this article, we show that blood markers of bone resorption inversely correlate with the amount of circulating Treg cells in healthy controls and rheumatoid arthritis patients, further suggesting that Treg cells may control bone destruction in vivo. Indeed, bone marrow from Foxp3-transgenic (Foxp3tg) mice fully protected human TNF transgenic (hTNFtg) mice from TNF-alpha-induced bone destruction, whereas Foxp3-deficient bone marrow enhanced local and systemic bone loss. The same protective effect was also obtained by treating hTNFtg mice with the CD28 superagonist mAb (CD28 SA), which increased Treg cell numbers. In both models, bone protection by Treg cells was associated with reduced osteoclast numbers, resulting in less bone-resorbing activity. Reduced osteoclast numbers were not caused by an intrinsic defect in osteoclast differentiation because osteoclast precursors from hTNFtg/Foxp3tg chimeras responded normally to M-CSF and receptor activator of NF-kappaB ligand. Although a decrease in the clinical signs of arthritis was observed in Foxp3tg bone marrow-transferred and CD28 SA-treated hTNFtg mice, the bone-protective effect of Treg cells was independent of the suppression of inflammation, as demonstrated by the increased systemic bone density observed in wild-type mice treated with CD28 SA. This work demonstrated that increasing Treg cell numbers improved clinical signs of arthritis and suppressed local and systemic bone destruction. Thus, enhancing the activity of Treg cells would be beneficial for the treatment of inflammation-induced bone loss observed in rheumatoid arthritis.

TNF and bone.

Bone is subject to permanent remodeling during development and through life. This activity is essential for (a) proper shaping and growth of each bone during development; (b) maintenance of bone mass as well as structural integrity of the micro architecture of bone through adult life, and (c) tissue repair needed for healing of fracture as well as of micro-damage. In addition to genetically linked rare developmental diseases, disturbances in bone remodeling are causing common bone pathologies, which severely impair the quality of life of patients. Among them are postmenopausal osteoporosis and local as well as systemic bone loss observed in chronic inflammatory diseases such as rheumatoid arthritis. The role of TNF-alpha in mediating bone remodeling will be presented and discussed in this chapter.

Increased bone density and resistance to ovariectomy-induced bone loss in FoxP3-transgenic mice based on impaired osteoclast differentiation.

OBJECTIVE: Immune activation triggers bone loss. Activated T cells are the cellular link between immune activation and bone destruction. The aim of this study was to determine whether immune regulatory mechanisms, such as naturally occurring Treg cells, also extend their protective effects to bone homeostasis in vivo. METHODS: Bone parameters in FoxP3-transgenic (Tg) mice were compared with those in their wild-type (WT) littermate controls. Ovariectomy was performed in FoxP3-Tg mice as a model of postmenopausal osteoporosis, and the bone parameters were analyzed. The bones of RAG-1(-/-) mice were analyzed following the adoptive transfer of isolated CD4+CD25+ T cells. CD4+CD25+ T cells and CD4+ T cells isolated from FoxP3-Tg mice and WT mice were cocultured with monocytes to determine their ability to suppress osteoclastogenesis in vitro. RESULTS: FoxP3-Tg mice developed higher bone mass and were protected from ovariectomy-induced bone loss. The increase in bone mass was found to be the result of impaired osteoclast differentiation and bone resorption in vivo. Bone formation was not affected. Adoptive transfer of CD4+CD25+ T cells into T cell-deficient RAG-1(-/-) mice also increased the bone mass, indicating that Treg cells directly affect bone homeostasis without the need to engage other T cell lineages. CONCLUSION: These data demonstrate that Treg cells can control bone resorption in vivo and can preserve bone mass during physiologic and pathologic bone remodeling.

R-spondin 1 protects against inflammatory bone damage during murine arthritis by modulating the Wnt pathway.

OBJECTIVE: During the course of different musculoskeletal diseases, joints are progressively damaged by inflammatory, infectious, or mechanical stressors, leading to joint destruction and disability. While effective strategies to inhibit joint inflammation, such as targeted cytokine-blocking therapy, have been developed during the last decade, the molecular mechanisms of joint damage are still poorly understood. This study was undertaken to investigate the role of the Wnt pathway modulator R-Spondin 1 (RSpo1) in protecting bone and cartilage in a mouse model of arthritis. METHODS: Tumor necrosis factor alpha (TNFalpha)-transgenic mice were treated with vehicle or Rspo1. Mice were evaluated for signs of arthritis, and histologic analysis of the hind paws was performed. Moreover, we determined the effect of Rspo1 on Wnt signaling activity and osteoprotegerin (OPG) expression in murine primary osteoblasts. RESULTS: The secreted Wnt pathway modulator RSpo1 was highly effective in preserving the structural integrity of joints in a TNFalpha-transgenic mouse model of arthritis by protecting bone and cartilage from inflammation-related damage. RSpo1 antagonized the Wnt inhibitor Dkk-1 and modulated Wnt signaling in mouse mesenchymal cells. In osteoblasts, RSpo1 induced differentiation and expression of OPG, thereby inhibiting osteoclastogenesis in vitro. In vivo, RSpo1 promoted osteoblast differentiation and bone formation while blocking osteoclast development, thereby contributing to the integrity of joints during inflammatory arthritis. CONCLUSION: Our results demonstrate the therapeutic potential of RSpo1 as an anabolic agent for the preservation of joint architecture.

The alpha-isoform of p38 MAPK specifically regulates arthritic bone loss.

Pharmacological inhibitors have provided evidence for the key role of p38 MAPK in osteoclast differentiation and in inflammation-induced bone loss. However, these inhibitors block more than one of the four p38 isoforms, usually p38alpha and p38beta, and sometimes also other kinases such as JNK3. We show in this study that p38alpha is the main p38 isoenzyme expressed in the osteoclast precursors and in the mature osteoclasts. p38alpha as well as its downstream substrates were phosphorylated in osteoclast progenitors stimulated by TNF-alpha. Using Mx-cre-mediated conditional gene inactivation we demonstrated that mice lacking p38alpha were protected against TNF-alpha-induced bone destruction at the site of inflammation as well as against TNF-alpha-mediated systemic bone loss. The bone protection was associated to decreased osteoclast numbers in vivo as well as a decreased IL-1beta expression in the inflamed tissue and in the isolated monocytes. The phenotype was cell autonomous because, similarly to p38alpha-deficient cells, knockdown of p38alpha in monocytes resulted in a decreased osteoclast differentiation in vitro. It was not caused by major changes in RANKL-mediated ERK or JNK activation but rather associated to an increased NF-kappaB activation caused by a decrease in IkappaBalpha recovery. Thus, our data show that developing specific inhibitors of the alpha-isoenzyme of p38 would be beneficial for the treatment of inflammation-induced bone destruction as observed in rheumatoid arthritis.

Osteoblast-specific inactivation of p53 results in locally increased bone formation.

Inactivation of the tumor suppressor p53 (encoded by the Trp53 gene) is relevant for development and growth of different cancers, including osteosarcoma, a primary bone tumor mostly affecting children and young adolescents. We have previously shown that deficiency of the ribosomal S6 kinase 2 (Rsk2) limits osteosarcoma growth in a transgenic mouse model overexpressing the proto-oncogene c-Fos. Our initial aim for the present study was to address the question, if Rsk2 deficiency would also influence osteosarcoma growth in another mouse model. For that purpose, we took advantage of Trp53fl/fl mice, which were crossed with Runx2Cre transgenic mice in order to inactivate p53 specifically in osteoblast lineage cells. However, since we unexpectedly identified Runx2Cre-mediated recombination also in the thymus, the majority of 6-month-old Trp53fl/fl;Runx2-Cre (thereafter termed Trp53Cre) animals displayed thymic lymphomas, similar to what has been described for Trp53-deficient mice. Since we did not detect osteosarcoma formation at that age, we could not follow our initial aim, but we studied the skeletal phenotype of Trp53Cre mice, with or without additional Rsk2 deficiency. Here we unexpectedly observed that Trp53Cre mice display a unique accumulation of trabecular bone in the midshaft region of the femur and the humerus, consistent with its previously established role as a negative regulator of osteoblastogenesis. Since this local bone mass increase in Trp53Cre mice was significantly reduced by Rsk2 deficiency, we isolated bone marrow cells from the different groups of mice and analyzed their behavior ex vivo. Here we observed a remarkable increase of colony formation, osteogenic differentiation and proliferation in Trp53Cre cultures, which was unaffected by Rsk2 deficiency. Our data thereby confirm a critical and tumorigenesis-independent function of p53 as a key regulator of mesenchymal cell differentiation.

JunD, not c-Jun, is the AP-1 transcription factor required for Ras-induced lung cancer.

The AP-1 transcription factor c-Jun is required for Ras-driven tumorigenesis in many tissues and is considered as a classical proto-oncogene. To determine the requirement for c-Jun in a mouse model of K-RasG12D-induced lung adenocarcinoma, we inducibly deleted c-Jun in the adult lung. Surprisingly, we found that inactivation of c-Jun, or mutation of its JNK phosphorylation sites, actually increased lung tumor burden. Mechanistically, we found that protein levels of the Jun family member JunD were increased in the absence of c-Jun. In c-Jun-deficient cells, JunD phosphorylation was increased, and expression of a dominant-active JNKK2-JNK1 transgene further increased lung tumor formation. Strikingly, deletion of JunD completely abolished Ras-driven lung tumorigenesis. This work identifies JunD, not c-Jun, as the crucial substrate of JNK signaling and oncogene required for Ras-induced lung cancer.

Mice Carrying a Ubiquitous R235W Mutation of Wnt1 Display a Bone-Specific Phenotype.

Since a key function of Wnt1 in brain development was established early on through the generation of non-viable Wnt1-deficient mice, it was initially surprising that WNT1 mutations were found to cause either early-onset osteoporosis (EOOP) or osteogenesis imperfecta type XV (OI-XV). The deduced function of Wnt1 as an osteoanabolic factor has been confirmed in various mouse models with bone-specific inactivation or overexpression, but mice carrying disease-causing Wnt1 mutations have not yet been described. Triggered by the clinical analysis of EOOP patients carrying a heterozygous WNT1 mutation (p.R235W), we introduced this mutation into the murine Wnt1 gene to address the question of whether this would cause a skeletal phenotype. We observed that Wnt1+/R235W and Wnt1R235W/R235W mice were born at the expected Mendelian ratio and that they did not display postnatal lethality or obvious nonskeletal phenotypes. At 12 weeks of age, the homozygous presence of the Wnt1 mutation was associated with reduced trabecular and cortical bone mass, explained by a lower bone formation rate compared with wild-type littermates. At 52 weeks of age, we also observed a moderate bone mass reduction in heterozygous Wnt1+/R235W mice, thereby underscoring their value as a model of WNT1-dependent EOOP. Importantly, when we treated wild-type and Wnt1+/R235W mice by daily injection of parathyroid hormone (PTH), we detected the same osteoanabolic influence in both groups, together with an increased cortical thickness in the mutant mice. Our data demonstrate the pathogenicity of the WNT1-R235W mutation, confirm that controlling skeletal integrity is the primary physiological function of Wnt1, and suggest that osteoanabolic treatment with teriparatide should be applicable for individuals with WNT1-dependent EOOP. © 2020 American Society for Bone and Mineral Research.

Osteoimmunology: a view from the bone.

Osteoimmunology can be defined in a very broad sense as the field of research focusing on interrelations between bone and the immune system. This is a rather opened field that covers at least three different issues. The first one is developmental, that is, organogenesis of the bones and immune systems. The second is post-developmental, that is, the role of the bone in the regulation of the immune response and role of the immune cells on the regulation of bone homeostasis. The third one is related to pathologies: Can immune cells be involved in the development of bone-related pathology? Can deregulation of the bone be causing immune-related diseases? I will not review in detail the bibliography covering osteoimmunology. This has been extensively done in Immunological Reviews (Vol. 208, December 2005) and Current Opinion in Rheumatology (Vol. 18, 2006). I will rather critically comment on hypotheses and concepts in osteoimmunology from a bone biologist's point of view.

Divergent off-target effects of RSK N-terminal and C-terminal kinase inhibitors in cardiac myocytes.

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of ß-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC50 for NA after D1870 pretreatment (DMSO/NA: 2.33 µmol/L vs. D1870/NA: 1.30 µmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.

Bone Marrow Plasma Cells Modulate Local Myeloid-Lineage Differentiation via IL-10.

Bone marrow plasma cells have been reported to represent a major source of IL-10; however, the impact of plasma cell derived IL-10 in that tissue remains poorly understood. We confirm in this study that even in the absence of acute immune reactions, mature plasma cells represent the dominant IL-10+ cell population in the bone marrow, and identify myeloid-lineage cells as a main local target for plasma cell derived IL-10. Using Vert-X IL-10 transcriptional reporter mice, we found that more than 50% of all IL-10+ cells in bone marrow were CD138+ plasma cells, while other IL-10+ B lineage cells were nearly absent in this organ. Accordingly, IL-10 was found in the supernatants of short-term cultures of FACS-sorted bone marrow plasma cells, confirming IL-10 secretion from these cells. IL-10+ bone marrow plasma cells showed a B220-/CD19-/MHCII low phenotype suggesting that these cells represent a mature differentiation stage. Approximately 5% of bone marrow leucocytes expressed the IL-10 receptor (IL-10R), most of them being CD115+/Ly6C+/CD11c- monocytes. Compared to littermate controls, young B lineage specific IL-10 KO mice showed increased numbers of CD115+ cells but normal populations of other myeloid cell types in bone marrow. However, at 7 months of age B lineage specific IL-10 KO mice exhibited increased populations of CD115+ myeloid and CD11c+ dendritic cells (DCs), and showed reduced F4/80 expression in this tissue; hence, indicating that bone marrow plasma cells modulate the differentiation of local myeloid lineage cells via IL-10, and that this effect increases with age. The effects of B cell/plasma cell derived IL-10 on the differentiation of CD115+, CD11c+, and F4/80+ myeloid cells were confirmed in co-culture experiments. Together, these data support the idea that IL-10 production is not limited to early plasma cell stages in peripheral tissues but is also an important feature of mature plasma cells in the bone marrow. Moreover, we provide evidence that already under homeostatic conditions in the absence of acute immune reactions, bone marrow plasma cells represent a non-redundant source for IL-10 that modulates local myeloid lineage differentiation. This is particularly relevant in older individuals.