Dampening of death pathways by schnurri-2 is essential for T-cell development.

Generation of a diverse and self-tolerant T-cell repertoire requires appropriate interpretation of T-cell antigen receptor (TCR) signals by CD4(+ ) CD8(+) double-positive thymocytes. Thymocyte cell fate is dictated by the nature of TCR-major-histocompatibility-complex (MHC)-peptide interactions, with signals of higher strength leading to death (negative selection) and signals of intermediate strength leading to differentiation (positive selection). Molecules that regulate T-cell development by modulating TCR signal strength have been described but components that specifically define the boundaries between positive and negative selection remain unknown. Here we show in mice that repression of TCR-induced death pathways is critical for proper interpretation of positive selecting signals in vivo, and identify schnurri-2 (Shn2; also known as Hivep2) as a crucial death dampener. Our results indicate that Shn2(-/-) double-positive thymocytes inappropriately undergo negative selection in response to positive selecting signals, thus leading to disrupted T-cell development. Shn2(-/-) double-positive thymocytes are more sensitive to TCR-induced death in vitro and die in response to positive selection interactions in vivo. However, Shn2-deficient thymocytes can be positively selected when TCR-induced death is genetically ablated. Shn2 levels increase after TCR stimulation, indicating that integration of multiple TCR-MHC-peptide interactions may fine-tune the death threshold. Mechanistically, Shn2 functions downstream of TCR proximal signalling compenents to dampen Bax activation and the mitochondrial death pathway. Our findings uncover a critical regulator of T-cell development that controls the balance between death and differentiation.

Control of bone resorption in mice by Schnurri-3.

Mice lacking the large zinc finger protein Schnurri-3 (Shn3) display increased bone mass, in part, attributable to augmented osteoblastic bone formation. Here, we show that in addition to regulating bone formation, Shn3 indirectly controls bone resorption by osteoclasts in vivo. Although Shn3 plays no cell-intrinsic role in osteoclasts, Shn3-deficient animals show decreased serum markers of bone turnover. Mesenchymal cells lacking Shn3 are defective in promoting osteoclastogenesis in response to selective stimuli, likely attributable to reduced expression of the key osteoclastogenic factor receptor activator of nuclear factor-κB ligand. The bone phenotype of Shn3-deficient mice becomes more pronounced with age, and mice lacking Shn3 are completely resistant to disuse osteopenia, a process that requires functional osteoclasts. Finally, selective deletion of Shn3 in the mesenchymal lineage recapitulates the high bone mass phenotype of global Shn3 KO mice, including reduced osteoclastic bone catabolism in vivo, indicating that Shn3 expression in mesenchymal cells directly controls osteoblastic bone formation and indirectly regulates osteoclastic bone resorption.

Emerging roles of PPARs in inflammation and immunity.

Lipids and lipid metabolism have well-documented regulatory effects on inflammatory processes. Recent work has highlighted the role of the peroxisome proliferator-activated receptors (PPARs)--a subset of the nuclear-hormone-receptor superfamily that are activated by various lipid species--in regulating inflammatory responses. Here, we describe how the PPARs, through their interactions with transcription factors and other cell-signalling systems, have important regulatory roles in innate and adaptive immunity.

Uncoupling of growth plate maturation and bone formation in mice lacking both Schnurri-2 and Schnurri-3.

Formation and remodeling of the skeleton relies on precise temporal and spatial regulation of genes expressed in cartilage and bone cells. Debilitating diseases of the skeletal system occur when mutations arise that disrupt these intricate genetic regulatory programs. Here, we report that mice bearing parallel null mutations in the adapter proteins Schnurri2 (Shn2) and Schnurri3 (Shn3) exhibit defects in patterning of the axial skeleton during embryogenesis. Postnatally, these compound mutant mice develop a unique osteochondrodysplasia. The deletion of Shn2 and Shn3 impairs growth plate maturation during endochondral ossification but simultaneously results in massively elevated trabecular bone formation. Hence, growth plate maturation and bone formation can be uncoupled under certain circumstances. These unexpected findings demonstrate that both unique and redundant functions reside in the Schnurri protein family that are required for proper skeletal patterning and remodeling.

Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice.

Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals.

Regulation of bone formation and immune cell development by Schnurri proteins.

Although identified over a decade ago, the function and physiological significance of the mammalian Schnurri protein family remained largely unknown. However, the recent generation and characterization of mice bearing null mutations in the individual Schnurri genes has led to the discovery of unexpected yet central roles for these large zinc-finger proteins in several biological processes. Here, we review findings of these studies and discuss the importance of the Schnurri protein family in regulating both the immune and skeletal systems.

Biomechanical and Bone Material Properties of Schnurri-3 Null Mice.

Schnurri-3 (Shn3) is an essential regulator of postnatal skeletal remodeling. Shn3-deficient mice (Shn3-/-) have high bone mass; however, their bone mechanical and material properties have not been investigated to date. We performed three-point bending of femora, compression tests of L3 vertebrae. We also measured intrinsic material properties, including bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics by quantitative backscatter electron imaging, as well as collagen cross-linking by Fourier transform infrared microspectroscopy of femora from Shn3-/- and WT mice at different ages (6 weeks, 4 months, and 18 months). Moreover, computer modeling was performed for the interpretation of the BMDD outcomes. Femora and L3 vertebrae from Shn3-/- aged 6 weeks revealed increased ultimate force (2.2- and 3.2-fold, p < .01, respectively). Mineralized bone volume at the distal femoral metaphysis was about twofold (at 6 weeks) to eightfold (at 4 and 18 months of age) in Shn3-/- (p < .001). Compared with WT, the average degree of trabecular bone mineralization was similar at 6 weeks, but increased at 4 and 18 months of age (+12.6% and +7.7%, p < .01, respectively) in Shn3-/-. The analysis of OLS characteristics revealed a higher OLS area for Shn3-/- versus WT at all ages (+16%, +23%, +21%, respectively, p < .01). The collagen cross-link ratio was similar between groups. We conclude that femora and vertebrae from Shn3-/- had higher ultimate force in mechanical testing. Computer modeling demonstrated that in cases of highly increased bone volume, the average degree of bone matrix mineralization can be higher than in WT bone, which was actually measured in the older Shn3-/- groups. The area of 2D osteocyte lacunae sections was also increased in Shn3-deficiency, which could only partly be explained by larger remnant areas of primary cortical bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Lentivirus delivery of shRNA constructs into osteoblasts.

Osteoblasts are the sole cell responsible for bone formation in vivo (1). Although genetic techniques have been extremely valuable to study the functions of certain genes in these cells in vivo, this approach is time consuming and expensive. An alternative loss-of-function approach that has been validated in many mammalian systems is shRNA-mediated gene silencing. This chapter describes methodology designed to introduce shRNA constructs into primary murine osteoblasts ex vivo in order to quickly assess the function of genes in osteoblast differentiation and extra cellular matrix mineralization. Both the production of shRNA-expressing lentiviruses and the infection of calvarial osteoblasts with these lentiviruses are detailed.

Control of postnatal bone mass by the zinc finger adapter protein Schnurri-3.

The completed skeleton undergoes continuous remodeling for the duration of adult life. Rates of bone formation by osteoblasts and bone resorption by osteoclasts determine adult bone mass. Abnormalities in either the osteoblast or osteoclast compartment affect bone mass and result in skeletal disorders, the most common of which is osteoporosis, a state of low bone mass. Much is known about the molecular control of bone formation and resorption from rare single gene disorders resulting in elevated or reduced bone mass. Such genetic disorders can be attributed either to osteoclast deficiencies, collectively termed "osteopetrosis," or to intrinsically elevated osteoblast activity, termed "osteosclerosis." However, an increasing need for anabolic therapies to prevent age-induced bone loss has stimulated a search for additional genes that act at the level of the osteoblast to regulate matrix synthesis. Recently, we have discovered a zinc finger adaptor protein called Schnurri-3 (Shn3) that potently regulates adult bone mass. Mice that lack Shn3 have normal skeletal morphogenesis but display profoundly elevated bone mass that increases with age. The molecular mechanism was revealed to be the recruitment of WWP1, a Nedd4 family E3 ubiquitin ligase, by Shn3 to the major transcriptional regulator of the osteoblast, Runx2. In the absence of Shn3, Runx2 degradation by WWP1 is inhibited resulting in increased levels of Runx2 protein and enhanced expression of Runx2 target genes leading to increased osteoblast synthetic activity. Small molecules that inhibit Shn3 or WWP1 may be attractive candidates for the treatment of diseases of low bone mass.

Schnurri-3: a key regulator of postnatal skeletal remodeling.

Schnurri-3, a large zinc finger protein distantly related to Drosophila Shn, is a potent and essential regulator of adult bone formation. Mice lacking Shn3 display an osteosclerotic phenotype with profoundly increased bone mass due to augmented osteoblast activity. Shn3 controls protein levels of Runx2, the principal regulator of osteoblast differentiation, by promoting its degradation. In osteoblasts, Shn3 functions as a component of a trimeric complex between Runx2 and the E3 ubiquitin ligase WWP1. This complex inhibits Runx2 function and expression of genes involved in extracellular matrix mineralization due to the ability of WWP1 to promote Runx2 polyubiquitination and proteasome-dependent degradation. Our study reveals an essential role for Shn3 as a regulator of postnatal bone mass. Compounds designed to block Shn3/WWP1 function may be possible therapeutic agents for the treatment of osteoporosis.