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Osteoclasts are bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathological bone erosion. Macrophage colony stimulating factor (M-CSF) is abundant in rheumatoid arthritis (RA). However, the role of M-CSF in arthritic bone erosion is not completely understood. Here, we show that M-CSF can promote osteoclastogenesis by triggering the proteolysis of c-FMS, a receptor for M-CSF, leading to the generation of FMS intracellular domain (FICD) fragments. Increased levels of FICD fragments positively regulated osteoclastogenesis but had no effect on inflammatory responses. Moreover, myeloid cell-specific FICD expression in mice resulted in significantly increased osteoclast-mediated bone resorption in an inflammatory arthritis model. The FICD formed a complex with DAP5, and the FICD/DAP5 axis promoted osteoclast differentiation by activating the MNK1/2/EIF4E pathway and enhancing NFATc1 protein expression. Moreover, targeting the MNK1/2 pathway diminished arthritic bone erosion. These results identified a novel role of c-FMS proteolysis in osteoclastogenesis and the pathogenesis of arthritic bone erosion.

Intervertebral disc degeneration and associated back pain are relatively common but sparsely understood conditions, affecting over 70% of the population during some point of life. Disc degeneration is often associated with a loss of nucleus pulposus (NP) cells. Genetic mouse models offer convenient avenues to understand the cellular and molecular regulation of the disc during its formation, growth, maintenance, and aging. However, due to the lack of inducible driver lines to precisely target NP cells in the postnatal mouse disc, progress in this area of research has been moderate. NP cells are known to express cytokeratin 19 (Krt19), and tamoxifen (Tam)‐inducible Krt19 CreERT allele is available. The current study describes the characterization of Krt19 CreERT allele to specifically and efficiently target NP cells in neonatal, skeletally mature, middle‐aged, and aged mice using two independent fluorescent reporter lines. The efficiency of recombination at all ages was validated by immunostaining for KRT19. Results show that following Tam induction, Krt19 CreERT specifically drives recombination of NP cells in the spine of neonatal and aged mice, while no recombination was detected in the surrounding tissues. Knee joints from skeletally mature Tam‐treated Krt19 CreERT/+; R26 tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19 CreERT allele for genetic characterization of NP cells at different stages of the mouse life., An inducible driver line to precisely target nucleus pulposus (NP) cells during the postnatal is required to understand the role of these cells during development and aging. The current study describes the characterization of Krt19CreERT allele to efficiently target NP cells in neonatal, skeletally mature, middle‐aged, and aged mice using two independent fluorescent reporter lines. Moreover, this allele did not target the surrounding cells in the spine or any cell in the knee joint, validating its specificity to target NP cells in the postnatal mouse.

Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (AF), and a pair of end plate (EP) that connects the disc to the vertebral bodies. Understanding the molecular and cellular basis of intervertebral disc growth, health, and aging will generate significant information for developing therapeutic approaches. Rapid and efficient preparations of homogeneous and pure cells are crucial for meaningful and rigorous downstream analysis at the cellular, molecular, and biochemical level. Cross‐sample contamination may influence the interpretation of the results. In addition to altering gene expression, slow or delayed isolation procedures will also cause the degradation of cells and biomolecules that create a bias in the outcomes of the study. The mouse model system is extensively used to understand the intervertebral disc biology. Here we describe two protocols: (a) for efficient isolation of pure NP, AF, and EP cells from mouse lumbar intervertebral disc. We validated the purity of the NP and AF cells using ShhCre/+; R26mT/mG/+ dual‐fluorescent reporter mice where all NP cells are GPF+ve, and by the sensitive approach of qPCR analysis using TaqMan probes for Shh, and Brachyury as NP‐specific markers, Tenomodulin as AF‐specific marker, and Osteocalcin as bone‐specific marker. (b) For isolation of high‐quality intact RNA with RIN of 9.3 to 10 from disc cells. These methods will be useful for the rigorous analysis of NP and AF cells, and improve our understanding of intervertebral disc biology.

Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.

Intervertebral discs are cartilaginous joints present between vertebrae. The centers of the intervertebral discs consist of a gelatinous nucleus pulposus derived from the embryonic notochord. With age or injury, intervertebral discs may degenerate, causing neurological symptoms including back pain, which affects millions of people worldwide. Back pain is a multifactorial disorder, and disc degeneration is one of the primary contributing factors. Recent studies in mice have identified the key molecules involved in the formation of intervertebral discs. Several of these key molecules including sonic hedgehog and Brachyury are not only expressed by notochord during development, but are also expressed by neonatal mouse nucleus pulposus cells, and are crucial for postnatal disc maintenance. These findings suggest that intrinsic signals in each disc may maintain the nucleus pulposus microenvironment. However, since expression of these developmental signals declines with age and degeneration, disc degeneration may be related to the loss of these intrinsic signals. In addition, findings from mouse and other mammalian models have identified similarities between the patterning capabilities of the embryonic notochord and young nucleus pulposus cells, suggesting that mouse is a suitable model system to understand disc development and aging. Future research aimed at understanding the upstream regulators of these developmental signals and the modes by which they regulate disc growth and maintenance will likely provide mechanistic insights into disc growth and aging. Further, such findings will likely provide insights relevant to the development of effective therapies for treatment of back pain and reversing the disc degenerative process. This article is categorized under: Birth Defects > Organ Anomalies Vertebrate Organogenesis > Musculoskeletal and Vascular Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Aging.

STUDY DESIGN Vertebral growth plates at different postnatal ages were assessed for active intercellular signaling pathways. OBJECTIVE To generate a spatial and temporal map of the major signaling pathways active in the postnatal mouse lumbar vertebral growth plate. SUMMARY OF BACKGROUND DATA The growth of all long bones is known to occur by cartilaginous growth plates. The growth plate is composed of layers of chondrocyets that actively proliferate, differentiate, die and, are replaced by bone. The role of major cell signaling pathways has been suggested for regulation of the fetal long bones. But not much is known about the molecular or cellular signals that control the postnatal vertebral growth plate and hence postnatal vertebral bone growth. Understanding such molecular mechanisms will help design therapeutic treatments for vertebral growth disorders such as scoliosis. METHODS Antibodies against activated downstream intermediates were used to identify cells in the growth plate responding to BMP, TGFβ, and FGF in cryosections of lumbar vertebrae from different postnatal age mice to identify the zones that were responding to these signals. Reporter mice were used to identify the chondrocytes responding to hedgehog (Ihh), and Wnt signaling. RESULTS We present a spatial/temporal map of these signaling pathways during growth, and differentiation of the mouse lumbar vertebral growth plate. CONCLUSION During growth and differentiation of the vertebral growth plate, its different components respond at different times to different intercellular signaling ligands. Response to most of these signals is dramatically downregulated at the end of vertebral growth.

FSH receptor has been shown to be specifically expressed only in the Sertoli cells in males. In one of our studies that consisted of deprival of endogenous FSH in immature rats and adult bonnet monkeys, atrophy of the epididymis was observed, cauda region being the most affected. Although epididymis is an androgen-dependent tissue, the changes in histology of the cauda region were observed without any associated change in the levels of testosterone in FSH-deprived animals. Considering this, it was of interest to evaluate the possibility of epididymis being a direct target for FSH action. In the present study, we have examined the expression of FSH receptor in the epididymis of rat and monkey. In the cauda region of rat epididymis, FSH receptor expression was demonstrated by RT-PCR and Northern and Western blot analyses. FSH receptor was found to be functional as observed by its ability to bind 125IoFSH, by an increase in cAMP production, and by BrdU incorporation following addition of FSH under in vitro conditions. These results suggest the possibility of a role for FSH in regulating the growth of the epididymis.

Intervertebral discs (IVD) are essential components of the vertebral column. They maintain separation, and provide shock absorbing buffers, between adjacent vertebrae, while also allowing movements between them. Each IVD consists of a central semi-liquid nucleus pulposus (NP) surrounded by a multi-layered fibrocartilagenous annulus fibrosus (AF). Although the IVDs grow and differentiate after birth along with the vertebral column, little is known about the mechanism of this. Understanding the signals that control normal IVD growth and differentiation would also provide potential therapies for degenerative disc disease, which is the major cause of lower back pain and affects a large proportion of the population. In this work, we show that during postnatal growth of the mouse, Sonic hedgehog (Shh) signaling from the NP cells controls many aspects of growth and differentiation of both the NP cells themselves and of the surrounding AF, and that it acts, at least partly, by regulating other signaling pathways in the NP and AF. Recent studies have shown that the NP cells arise from the embryonic notochord, which acts as a major signaling center in the embryo. This work shows that this notochord-derived tissue continues to carry out a major signaling function in the postnatal body and that the IVDs are signaling centers, in addition to their already known functions in the mechanics of vertebral column function.

STUDY DESIGN Intervertebral discs at different postnatal ages were assessed for active intercellular signaling pathways. OBJECTIVE To generate a spatial and temporal map of the signaling pathways active in the postnatal intervertebral disc (IVD). SUMMARY OF BACKGROUND DATA The postnatal IVD is a complex structure, consisting of 3 histologically distinct components, the nucleus pulposus, fibrous anulus fibrosus, and endplate. These differentiate and grow during the first 9 weeks of age in the mouse. Identification of the major signaling pathways active during and after the growth and differentiation period will allow functional analysis using mouse genetics and identify targets for therapy for individual components of the disc. METHODS Antibodies specific for individual cell signaling pathways were used on cryostat sections of IVD at different postnatal ages to identify which components of the IVD were responding to major classes of intercellular signal, including sonic hedgehog, Wnt, TGFbeta, FGF, and BMPs. RESULTS We present a spatial/temporal map of these signaling pathways during growth, differentiation, and aging of the disc. CONCLUSION During growth and differentiation of the disc, its different components respond at different times to different intercellular signaling ligands. Most of these are dramatically downregulated at the end of disc growth.

Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease.

Introduction: Degenerative disc disease is a major cause of lower back pain. However the molecular signals that control normal growth and differentiation of the disc are not well defined. We hypothesize that nucleus pulposus (NP) cells, which originate from the embryonic notochord, provide signals that control growth and differentiation of the postnatal disc. Methods: Postnatal day 4 mouse IVDs were cultured in serum-free DMEM Ham/F12 medium at 37 °C in 5% CO2 for 2–5 days on type IV collagen-coated cell culture inserts. Cyclopamine (250 μM) was used as a Shh antagonist, and its specificity was tested by add-back experiments using 100 nM recombinant Shh (rShh). Cell proliferation was assayed using pulselabelling with BrdU. 6 μm thick cryosections were immunostained (IHC) using antibodies specific to IVD differentiation markers, and Cy5 conjugated secondary antibodies. Imaging was carried out using confocal microscope. To test the role of Shh in vivo, triple transgenic mice [(tetO)7CMV-cre:R26 rtTA:Shhflx/flx] were injected with 2 μg of doxycycline on postnatal day 4 to conditionally delete Shh, and the lumbar spine was collected 5 days later. Results were compared to vehicle treated controls. Results: Both cyclopamine treatment in vitro and conditional Shh knockout in vivo caused rounding up and aggregation of the NP cells, and loss of orientation of the layers of the annulus fibrosus (AF). There was also a loss of expression of both NP and AF differentiation markers. BrdU incorporation studies showed loss of proliferation of NP cells. IHC for activated caspase-3 showed increased levels of apoptosis of NP cells rShh replacement and reversed the cyclopamine phenotype in vitro. Conclusions: These results show that Shh is essential for postnatal growth and differentiation of both the NP and AF. Significance: Identification of signals controlling IVD growth and differentiation offers the opportunity for the development of biological repair as an alternative to surgery.