Mei Wan

Disruption of LRP6 in osteoblasts blunts the bone anabolic activity of PTH.

Mutations in low‐density lipoprotein receptor‐related protein 6 (LRP6) are associated with human skeletal disorders. LRP6 is required for parathyroid hormone (PTH)‐stimulated signaling pathways in osteoblasts. We investigated whether LRP6 in osteoblasts directly regulates bone remodeling and mediates the bone anabolic effects of PTH by specifically deleting LRP6 in mature osteoblasts in mice (LRP6 KO). Three‐month‐old LRP6 KO mice had a significant reduction in bone mass in the femora secondary spongiosa relative to their wild‐type littermates, whereas marginal changes were found in femoral tissue of 1‐month‐old LRP6 KO mice. The remodeling area of the 3‐month‐old LRP6 KO mice showed a decreased bone formation rate as detected by Goldners Trichrome staining and calcein double labeling. Bone histomorphometric and immumohistochemical analysis revealed a reduction in osteoblasts but little change in the numbers of osteoclasts and osteoprogenitors/osteoblast precursors in LRP6 KO mice compared with wild‐type littermates. In addition, the percentage of the apoptotic osteoblasts on the bone surface was higher in LRP6 KO mice compared with wild‐type littermates. Intermittent injection of PTH had no effect on bone mass or osteoblastic bone formation in either trabecular and cortical bone in LRP6 KO mice, whereas all were enhanced in wild‐type littermates. Additionally, the anti‐apoptotic effect of PTH on osteoblasts in LRP6 KO mice was less significant compared with wild‐type mice. Therefore, our findings demonstrate that LRP6 in osteoblasts is essential for osteoblastic differentiation during bone remodeling and the anabolic effects of PTH.

Functional Effects of TGF-β1 on Mesenchymal Stem Cell Mobilization in Cockroach Allergen–Induced Asthma

Mesenchymal stem cells (MSCs) have been suggested to participate in immune regulation and airway repair/remodeling. TGF-β1 is critical in the recruitment of stem/progenitor cells for tissue repair, remodeling, and cell differentiation. In this study, we sought to investigate the role of TGF-β1 in MSC migration in allergic asthma. We examined nestin expression (a marker for MSCs) and TGF-β1 signaling activation in airways in cockroach allergen extract (CRE)–induced mouse models. Compared with control mice, there were increased nestin+ cells in airways and higher levels of active TGF-β1 in serum and p-Smad2/3 expression in lungs of CRE-treated mice. Increased activation of TGF-β1 signaling was also found in CRE-treated MSCs. We then assessed MSC migration induced by conditioned medium from CRE-challenged human epithelium in air/liquid interface culture in Transwell assays. MSC migration was stimulated by epithelial-conditioned medium, but was significantly inhibited by either TGF-β1–neutralizing Ab or TβR1 inhibitor. Intriguingly, increased migration of MSCs from blood and bone marrow to the airway was also observed after systemic injection of GFP+ MSCs and from bone marrow of Nes-GFP mice following CRE challenge. Furthermore, TGF-β1–neutralizing Ab inhibited the CRE-induced MSC recruitment, but promoted airway inflammation. Finally, we investigated the role of MSCs in modulating CRE-induced T cell response and found that MSCs significantly inhibited CRE-induced inflammatory cytokine secretion (IL-4, IL-13, IL-17, and IFN-γ) by CD4+ T cells. These results suggest that TGF-β1 may be a key promigratory factor in recruiting MSCs to the airways in mouse models of asthma.

Lipoprotein receptor–related protein 6 is required for parathyroid hormone–induced Sost suppression

Parathyroid hormone (PTH) suppresses the expression of the bone formation inhibitor sclerostin (Sost) in osteocytes by inducing nuclear accumulation of histone deacetylases (HDACs) to inhibit the myocyte enhancer factor 2 (MEF2)‐dependent Sost bone enhancer. Previous studies revealed that lipoprotein receptor–related protein 6 (LRP6) mediates the intracellular signaling activation and the anabolic bone effect of PTH. Here, we investigated whether LRP6 mediates the inhibitory effect of PTH on Sost using an osteoblast‐specific Lrp6‐knockout (LRP6‐KO) mouse model. An increased level of Sost mRNA expression was detected in femur tissue from LRP6‐KO mice, compared to wild‐type littermates. The number of osteocytes expressing sclerostin protein was also increased in bone tissue of LRP6‐KO littermates, indicating a negative regulatory role of LRP6 on Sost/sclerostin. In wild‐type littermates, intermittent PTH treatment significantly suppressed Sost mRNA expression in bone and the number of sclerostin+ osteocytes, while the effect of PTH was much less significant in LRP6‐KO mice. Additionally, PTH‐induced downregulation of MEF2C and 2D, as well as HDAC changes in osteocytes, were abrogated in LRP6‐KO mice. These data indicate that LRP6 is required for PTH suppression of Sost expression.

Aryl Hydrocarbon Receptor Protects Lungs from Cockroach Allergen–Induced Inflammation by Modulating Mesenchymal Stem Cells

Exposure to cockroach allergen leads to allergic sensitization and increased risk of developing asthma. Aryl hydrocarbon receptor (AhR), a receptor for many common environmental contaminants, can sense not only environmental pollutants but also microbial insults. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity to modulate immune responses. In this study, we investigated whether AhR can sense cockroach allergens and modulate allergen-induced lung inflammation through MSCs. We found that cockroach allergen–treated AhR-deficient (AhR−/−) mice showed exacerbation of lung inflammation when compared with wild-type (WT) mice. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AhR agonist, significantly suppressed allergen-induced mouse lung inflammation. MSCs were significantly reduced in cockroach allergen–challenged AhR−/− mice as compared with WT mice, but increased in cockroach allergen–challenged WT mice when treated with TCDD. Moreover, MSCs express AhR, and AhR signaling can be activated by cockroach allergen with increased expression of its downstream genes cyp1a1 and cyp1b1. Furthermore, we tracked the migration of i.v.-injected GFP+ MSCs and found that cockroach allergen–challenged AhR−/− mice displayed less migration of MSCs to the lungs compared with WT. The AhR-mediated MSC migration was further verified by an in vitro Transwell migration assay. Epithelial conditioned medium prepared from cockroach extract–challenged epithelial cells significantly induced MSC migration, which was further enhanced by TCDD. The administration of MSCs significantly attenuated cockroach allergen–induced inflammation, which was abolished by TGF-β1–neutralizing Ab. These results suggest that AhR plays an important role in protecting lungs from allergen-induced inflammation by modulating MSC recruitment and their immune-suppressive activity.

LRP6 in mesenchymal stem cells is required for bone formation during bone growth and bone remodeling

Lipoprotein receptor-related protein 6 (LRP6) plays a critical role in skeletal development and homeostasis in adults. However, the role of LRP6 in mesenchymal stem cells (MSCs), skeletal stem cells that give rise to osteoblastic lineage, is unknown. In this study, we generated mice lacking LRP6 expression specifically in nestin+ MSCs by crossing nestin-Cre mice with LRP6flox mice and investigated the functional changes of bone marrow MSCs and skeletal alterations. Mice with LRP6 deletion in nestin+ cells demonstrated reductions in body weight and body length at 1 and 3 months of age. Bone architecture measured by microCT (µCT) showed a significant reduction in bone mass in both trabecular and cortical bone of homozygous and heterozygous LRP6 mutant mice. A dramatic reduction in the numbers of osteoblasts but much less significant reduction in the numbers of osteoclasts was observed in the mutant mice. Osterix+ osteoprogenitors and osteocalcin+ osteoblasts significantly reduced at the secondary spongiosa area, but only moderately decreased at the primary spongiosa area in mutant mice. Bone marrow MSCs from the mutant mice showed decreased colony forming, cell viability and cell proliferation. Thus, LRP6 in bone marrow MSCs is essential for their survival and proliferation, and therefore, is a key positive regulator for bone formation during skeletal growth and remodeling.

Programmed cell senescence in skeleton during late puberty

Mesenchymal stem/progenitor cells (MSPCs) undergo rapid self-renewal and differentiation, contributing to fast skeletal growth during childhood and puberty. It remains unclear whether these cells change their properties during late puberty to young adulthood, when bone growth and accrual decelerate. Here we show that MSPCs in primary spongiosa of long bone in mice at late puberty undergo normal programmed senescence, characterized by loss of nestin expression. MSPC senescence is epigenetically controlled by the polycomb histone methyltransferase enhancer of zeste homolog 2 (Ezh2) and its trimethylation of histone H3 on Lysine 27 (H3K27me3) mark. Ezh2 maintains the repression of key cell senescence inducer genes through H3K27me3, and deletion of Ezh2 in early pubertal mice results in premature cellular senescence, depleted MSPCs pool, and impaired osteogenesis as well as osteoporosis in later life. Our data reveals a programmed cell fate change in postnatal skeleton and unravels a regulatory mechanism underlying this phenomenon.Mesenchymal stem cells are essential for bone development, but it is unclear if their activity is maintained after late puberty, when bone growth decelerates. The authors show that during late puberty in mice, these cells undergo senescence under the epigenetic control of Ezh2.

Mannose receptor modulates macrophage polarization and allergic inflammation through miR-511-3p

&NA; Figure. No caption available. Background: Mannose receptor (MRC1/CD206) has been suggested to mediate allergic sensitization and asthma to multiple glycoallergens, including cockroach allergens. Objective: We sought to determine the existence of a protective mechanism through which MRC1 limits allergic inflammation through its intronic miR‐511‐3p. Methods: We examined MRC1‐mediated cockroach allergen uptake by lung macrophages and lung inflammation using C57BL/6 wild‐type (WT) and Mrc1−/− mice. The role of miR‐511‐3p in macrophage polarization and cockroach allergen–induced lung inflammation in mice transfected with adeno‐associated virus (AAV)–miR‐511‐3p (AAV–cytomegalovirus–miR‐511‐3p–enhanced green fluorescent protein) was analyzed. Gene profiling of macrophages with or without miR‐511‐3p overexpression was also performed. Results: Mrc1−/− lung macrophages showed a significant reduction in cockroach allergen uptake compared with WT mice, and Mrc1−/− mice had an exacerbated lung inflammation with increased levels of cockroach allergen–specific IgE and TH2/TH17 cytokines in a cockroach allergen–induced mouse model compared with WT mice. Macrophages from Mrc1−/− mice showed significantly reduced levels of miR‐511‐3 and an M1 phenotype, whereas overexpression of miR‐511‐3p rendered macrophages to exhibit a M2 phenotype. Furthermore, mice transfected with AAV–miR‐511‐3p showed a significant reduction in cockroach allergen–induced inflammation. Profiling of macrophages with or without miR‐511‐3p overexpression identified 729 differentially expressed genes, wherein expression of prostaglandin D2 synthase (Ptgds) and its product PGD2 were significantly downregulated by miR‐511‐3p. Ptgds showed a robust binding to miR‐511‐3p, which might contribute to the protective effect of miR‐511‐3p. Plasma levels of miR‐511‐3p were significantly lower in human asthmatic patients compared with nonasthmatic subjects. Conclusion: These studies support a critical but previously unrecognized role of MRC1 and miR‐511‐3p in protection against allergen‐induced lung inflammation.

Ras homolog family member A/Rho-associated protein kinase 1 signaling modulates lineage commitment of mesenchymal stem cells in asthmatic patients through lymphoid enhancer–binding factor 1

Background Numbers of mesenchymal stem cells (MSCs) are increased in the airways after allergen challenge. Ras homolog family member A (RhoA)/Rho‐associated protein kinase 1 (ROCK) signaling is critical in determining the lineage fate of MSCs in tissue repair/remodeling. Objectives We sought to investigate the role of RhoA/ROCK signaling in lineage commitment of MSCs during allergen‐induced airway remodeling and delineate the underlying mechanisms. Methods Active RhoA expression in lung tissues of asthmatic patients and its role in cockroach allergen–induced airway inflammation and remodeling were investigated. RhoA/ROCK signaling–mediated MSC lineage commitment was assessed in an asthma mouse model by using MSC lineage tracing mice (nestin‐Cre; ROSA26‐EYFP). The role of RhoA/ROCK in MSC lineage commitment was also examined by using MSCs expressing constitutively active RhoA (RhoA‐L63) or dominant negative RhoA (RhoA‐N19). Downstream RhoA‐regulated genes were identified by using the Stem Cell Signaling Array. Results Lung tissues from asthmatic mice showed increased expression of active RhoA when compared with those from control mice. Inhibition of RhoA/ROCK signaling with fasudil, a RhoA/ROCK inhibitor, reversed established cockroach allergen–induced airway inflammation and remodeling, as assessed based on greater collagen deposition/fibrosis. Furthermore, fasudil inhibited MSC differentiation into fibroblasts/myofibroblasts but promoted MSC differentiation into epithelial cells in asthmatic nestin‐Cre; ROSA26‐EYFP mice. Consistently, expression of RhoA‐L63 facilitated differentiation of MSCs into fibroblasts/myofibroblasts, whereas expression of RhoA‐19 switched the differentiation toward epithelial cells. The gene array identified the Wnt signaling effector lymphoid enhancer–binding factor 1 (Lef1) as the most upregulated gene in RhoA‐L63–transfected MSCs. Knockdown of Lef1 induced MSC differentiation away from fibroblasts/myofibroblasts but toward epithelial cells. Conclusions These findings uncover a previously unrecognized role of RhoA/ROCK signaling in MSC‐involved airway repair/remodeling in the setting of asthma.

Oxidized phospholipids are ligands for LRP6

Low-density lipoprotein receptor–related protein 6 (LRP6) is a co-receptor for Wnt signaling and can be recruited by multiple growth factors/hormones to their receptors facilitating intracellular signaling activation. The ligands that bind directly to LRP6 have not been identified. Here, we report that bioactive oxidized phospholipids (oxPLs) are native ligands of LRP6, but not the closely related LRP5. oxPLs are products of lipid oxidation involving in pathological conditions such as hyperlipidemia, atherosclerosis, and inflammation. We found that cell surface LRP6 in bone marrow mesenchymal stromal cells (MSCs) decreased rapidly in response to increased oxPLs in marrow microenvironment. LRP6 directly bound and mediated the uptake of oxPLs by MSCs. oxPL-LRP6 binding induced LRP6 endocytosis through a clathrin-mediated pathway, decreasing responses of MSCs to osteogenic factors and diminishing osteoblast differentiation ability. Thus, LRP6 functions as a receptor and molecular target of oxPLs for their adverse effect on MSCs, revealing a potential mechanism underlying atherosclerosis-associated bone loss.Bone loss: revealing a molecular cause of ‘numb’ stem cellsA constituent of oxidized ‘bad cholesterol’ blocks essential signaling processes leading to pathogenic bone loss. LRP6 is a crucial receptor involved in multiple physiological processes, including bone loss; however, direct pathogenic modulators of the receptor have yet to be elucidated. Now, John Hopkins University School of Medicine’s Mei Wan, with a US and Chinese research team, has discovered that oxPL, a byproduct of the oxidization of cholesterol carrier LDL, binds directly to LRP6 and causes its removal from the surface of bone marrow stem cells. As a result, these stem cells are unable to sense the external signaling molecules that drive bone growth. oxPLs are products of diseases such as hyperlipidemia and atherosclerosis, and this paper helps to reveal their pathogenesis and offers potential targets for therapeutic interventions.

Ciliary parathyroid hormone signaling activates transforming growth factor-β to maintain intervertebral disc homeostasis during aging

Degenerative disc disease (DDD) is associated with intervertebral disc degeneration of spinal instability. Here, we report that the cilia of nucleus pulposus (NP) cells mediate mechanotransduction to maintain anabolic activity in the discs. We found that mechanical stress promotes transport of parathyroid hormone 1 receptor (PTH1R) to the cilia and enhances parathyroid hormone (PTH) signaling in NP cells. PTH induces transcription of integrin αvβ6 to activate the transforming growth factor (TGF)-β-connective tissue growth factor (CCN2)-matrix proteins signaling cascade. Intermittent injection of PTH (iPTH) effectively attenuates disc degeneration of aged mice by direct signaling through NP cells, specifically improving intervertebral disc height and volume by increasing levels of TGF-β activity, CCN2, and aggrecan. PTH1R is expressed in both mouse and human NP cells. Importantly, knockout PTH1R or cilia in the NP cells results in significant disc degeneration and blunts the effect of PTH on attenuation of aged discs. Thus, mechanical stress-induced transport of PTH1R to the cilia enhances PTH signaling, which helps maintain intervertebral disc homeostasis, particularly during aging, indicating therapeutic potential of iPTH for DDD.Degenerative disc disease: hormone signaling keeps intervertebral core in balanceSensory structures found in the jelly-like space between spinal discs release a hormone that helps preserve back health in aging mice. Xu Cao from Johns Hopkins University in Baltimore, Maryland, USA, and colleagues observed that levels of a critical growth factor declined in the space between adjacent vertebrae as mice aged, and that injecting a naturally occurring hormone that activates this growth factor could attenuate disc degeneration in older animals. The researchers showed, in response to mechanical stresses, receptor proteins that respond to this hormone relocate themselves to particular sensory organelles known as cilia that found within cells of the intervertebral core. That results in elevated hormone signaling—and drugs designed to have the same effect could help treat degenerative disc disease, one of the most common causes of chronic back pain.