Augmenting MNK1/2 activation by c-FMS proteolysis promotes osteoclastogenesis and arthritic bone erosion.

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.

Age-related molecular changes in the lumbar dorsal root ganglia of mice: Signs of sensitization, and inflammatory response.

Aging is a major risk factor for numerous painful, inflammatory, and degenerative diseases including disc degeneration. A better understanding of how the somatosensory nervous system adapts to the changing physiology of the aging body will be of great significance for our expanding aging population. Previously, we reported that chronological aging of mouse lumbar discs is pathological and associated with behavioral changes related to pain. It is established that with age and degeneration the lumbar discs become inflammatory and innervated. Here we analyze the aging lumbar dorsal root ganglia (DRGs) and spinal cord dorsal horn (SCDH) in mice between 3 and 24 months of age for age-related somatosensory adaptations. We observe that as mice age there are signs of peripheral sensitization, and response to inflammation at the molecular and cellular level in the DRGs. From 12 months onwards the mRNA expression of vasodilator and neurotransmitter, Calca (CGRP); stress (and survival) marker, Atf3; and neurotrophic factor, Bdnf, increases linearly with age in the DRGs. Further, while the mRNA expression of neuropeptide, Tac1, precursor of Substance P, did not change at the transcriptional level, TAC1 protein expression increased in 24-month-old DRGs. Additionally, elevated expression of NFκB subunits, Nfkb1 and Rela, but not inflammatory mediators, Tnf, Il6, Il1b, or Cox2, in the DRGs suggest peripheral nerves are responding to inflammation, but do not increase the expression of inflammatory mediators at the transcriptional level. These results identify a progressive, age-related shift in the molecular profile of the mouse somatosensory nervous system and implicates nociceptive sensitization and inflammatory response.

Characterization of Krt19 CreERT allele for targeting the nucleus pulposus cells in the postnatal mouse intervertebral disc.

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 Krt19CreERT allele is available. The current study describes the characterization of Krt19CreERT 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, Krt19CreERT 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 Krt19CreERT/+ ; R26tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19CreERT allele for genetic characterization of NP cells at different stages of the mouse life.

Chondrocyte-like nested cells in the aged intervertebral disc are late-stage nucleus pulposus cells.

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.

Aging of mouse intervertebral disc and association with back pain.

With the increased burden of low back pain (LBP) in our globally aging population there is a need to develop preclinical models of LBP that capture clinically relevant features of physiological aging, degeneration, and disability. Here we assess the validity of using a mouse model system for age-related LBP by characterizing aging mice for features of intervertebral disc (IVD) degeneration, molecular markers of peripheral sensitization, and behavioral signs of pain. Compared to three-month-old and one-year-old mice, two-year-old mice show features typical of IVD degeneration including loss of disc height, bulging, innervation and vascularization in the caudal lumbar IVDs. Aging is also associated with the loss of whole-body bone mineral density in both male and female mice, but not associated with percent lean mass or body fat. Additionally, two-year-old mice have an accumulation of TRPA1 channels and sodium channels NaV1.8 and NaV1.9 in the L4 and L5 lumbar dorsal root ganglia consistent with changes in nociceptive signaling. Lastly, the effect of age, sex, and weight on mobility, axial stretching and radiating pain measures was assessed in male and female mice ranging from two months to two years in a general linear model. The model revealed that regardless of sex or weight, increased age was a predictor of greater reluctance to perform axial stretching and sensitivity to cold, but not heat in mice.

Formation of the sacrum requires down-regulation of sonic hedgehog signaling in the sacral intervertebral discs.

In humans, the sacrum forms an important component of the pelvic arch, and it transfers the weight of the body to the lower limbs. The sacrum is formed by collapse of the intervertebral discs (IVDs) between the five sacral vertebrae during childhood, and their fusion to form a single bone. We show that collapse of the sacral discs in the mouse is associated with the down-regulation of sonic hedgehog (SHH) signaling in the nucleus pulposus (NP) of the disc, and many aspects of this phenotype can be reversed by experimental postnatal activation of hedgehog (HH) signaling. We have previously shown that SHH signaling is essential for the normal postnatal growth and differentiation of intervertebral discs elsewhere in the spine, and that loss of SHH signaling leads to pathological disc degeneration, a very common disorder of aging. Thus, loss of SHH is pathological in one region of the spine but part of normal development in another.

Role of Sonic Hedgehog Signaling Pathway in Intervertebral Disc Formation and Maintenance.

A PURPOSE OF REVIEW: The intervertebral discs (IVD) are an essential component of the spine. Degeneration of the discs, commonly due to age or injury, is a leading cause of chronic lower back pain. Despite its high prevalence, there is no effective treatment for disc disease due to limited understanding of disc at the cellular and molecular level. B RECENT FINDINGS: Recent research has demonstrated the importance of the intracellular developmental pathway sonic hedgehog (Shh) during the formation and postnatal maintenance of the IVD. Recent studies corroborate that the down-regulation of SHH expression is associated with pathological changes in the IVDs and demonstrate the reactivation of the hedgehog pathway as a promising avenue for rescuing health disc structure and function. C SUMMARY: Understanding the role of developmental signaling pathways that regulate disc formation and maintenance may help develop strategies to recapitulate the same mechanism for disc treatment and hence improve the quality and longevity of patient lives.

Wnt signaling activates Shh signaling in early postnatal intervertebral discs, and re-activates Shh signaling in old discs in the mouse.

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.

Shh signaling from the nucleus pulposus is required for the postnatal growth and differentiation of the mouse intervertebral disc.

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.

Intercellular signaling pathways active during and after growth and differentiation of the lumbar vertebral growth plate.

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, TGFß, 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.

Intercellular signaling pathways active during intervertebral disc growth, differentiation, and aging.

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.

Postnatal growth, differentiation, and aging of the mouse intervertebral disc.

STUDY DESIGN: This study follows postnatal intervertebral disc (IVD) growth and differentiation in the mouse. OBJECTIVE.: To initiate use of the mouse as a model system for postnatal IVD differentiation and growth, and to serve as a basis for assaying changes caused by disease or genetic or experimental perturbation. SUMMARY OF BACKGROUND DATA: Lower back pain caused by disc degeneration is one of the most common clinical conditions worldwide. There is currently no completely effective treatment, largely because of a lack of basic knowledge of the molecular and cellular controls of disc growth, differentiation, and maintenance after birth. METHODS: Conventional histology of decalcified IVDs, differential interference contrast, polarizing optics, immunocytochemistry, laser capture microscopy followed by molecular analysis of the dissected cells by reverse transcriptase polymerase chain reaction. RESULTS: There is a single postnatal growth spurt in the mouse IVD, between birth and 9 weeks of age. Cell proliferation was found in the nucleus pulposus (NP) and anulus fibrosus (AF) only until 3 weeks of age. Most of the postnatal growth of the IVD is due to accumulating extracellular matrix. NP cell numbers decline steadily after 2 weeks of age, because of apoptosis. Laser capture microscopy was used to dissect NP cells from the disc, and showed that these cells express markers of the embryonic notochord. The postnatal AF appears initially as a continuous structure surrounding the NP. This structure differentiates, during the first 2 postnatal weeks, to form the mineralized, but nonossified endplate over the surfaces of the vertebral growth plates, and the mature fibrous AF (fAF) passing between adjacent vertebrae. The fact that the mature fAF and the endplate form from an originally continuous layer of cells explains the anatomic relationship between these 2 structures, in which the fAF inserts into the vertebral endplate. CONCLUSION: Growth of the IVD takes place during the first 9 postnatal weeks, although cell proliferation ceases after 3 weeks. After birth, the early postnatal IVD differentiates into 3 tissue types, the NP, the fAF between the vertebrae, and the mineralized endplates over the surfaces of the vertebrae.

Effect of deprivation of endogenous follicle stimulating hormone on rat epididymis: a histological evaluation.

The growth and function of the epididymis are regulated by testosterone produced by Leydig cells in the testes. In the present study it was observed that neutralization of endogenous follicle stimulating hormone (FSH) in immature rats using a highly specific antiserum to ovine FSH resulted in changes in the histology of the epididymis along with a decrease (50-60%) in its weight compared with the normal serum-treated controls. These changes were observed in both rat and monkey epididymis without any decrease in serum testosterone, on which epididymis is known to be dependent. A detailed study was therefore carried out on the effects of deprivation of FSH or testosterone on the histology of epididymis. The changes in epididymal histology following FSH deprivation included a decrease in the size of the tubule lumen in the rat as well as in the adult male bonnet monkey in which the antiserum against ovine FSH was raised. Intensive vacuolization and uneven surface of the luminal epithelium was also observed. In contrast, the effect of deprivation of testosterone support by way of administration of LH antiserum or fiutamide resulted in a decrease in the size of the lumen and degenerative changes. These results suggest that cauda epididymidis is a target for FSH action.

Demonstration of follicle-stimulating hormone receptor in cauda epididymis of rat.

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.

Regulation of FSH receptor, PKIbeta, IL-6 and calcium mobilization: Possible mediators of differential action of FSH.

Sertoli cells support the development of germ cells by providing a microenvironment in the seminiferous tubules. FSH stimulates Sertoli cell proliferation only during neonatal period till day 18 in the immature rat whereas FSH regulates only functional parameters in the adult rat Sertoli cells. This suggests that FSH exerts differential action in immature and adult Sertoli cells. In an attempt to elucidate the mechanism by which FSH exerts the differential effects, we have carried out both in vivo and in vitro studies using Sertoli cells isolated from immature (7-10 days old) and adult (90 days old) rats. The differential role of FSH was studied at the receptor as well as at the signaling level. Monitoring the level of expression of FSH receptor by RTPCR and northern blot analysis revealed that the expression was more in immature Sertoli cells. Furthermore, it was found that FSH up (1.8-fold) regulates its receptor level only in the immature Sertoli cells and not in the adult. Results also revealed that PKIbeta and calcium, which are the downstream signaling molecules, are involved in FSH regulated Sertoli cells proliferation. It was also observed that FSH up (1.4-fold) regulates the levels of expression of IL-6 mRNA only in the immature rat Sertoli cells suggesting the possibility of its involvement in FSH regulated Sertoli cell proliferation.