Genetic Regulation of the Growth Plate

The epiphyseal growth plate consists of a layer of cartilage present only during the growth period and vanishes soon after puberty in long bones. It is divided to three well-defined zones, from epiphyses; resting, proliferative, and hypertrophic zones. Chondrocyte proliferation and differentiation and subsequent bone formation in this cartilage are controlled by various endocrine, autocrine, and paracrine factors which finally results into elimination of the cartilaginous tissue and promotion of the epiphyseal fusion. As chondrocytes differentiate from round, quiescent, and single structure to flatten and proliferative and then large and terminally differentiated, they experience changes in their gene expression pattern which allow them to transform from cartilaginous tissue to bone. This review summarizes the literature in this area and shortly describes different factors that affect growth plate cartilage both at the local and systemic levels. This may eventually help us to develop new treatment strategies of different growth disorders

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale

Effect of estrogen on longitudinal bone growth

Longitudinal bone growth occurs at the growth plate, a thin layer of cartilage between the epiphysis and the metaphysis of long bones. In the growth plate resting chondrocytes proliferate, differentiate into a hypertrophic form, and finally become terminal hypertrophic chondrocytes before giving rise to bone. Estrogen is essential for skeIetal growth. Extreme early puberty often leads to short stature, whereas significantly delayed puberty leads to increased final stature. Moreover, high doses of estrogen were used to prevent extreme tall stature in girls, but the treatment was associated with severe side effects. The effect of estrogen is mediated via two known estrogen receptors, ERalpha and ERbeta that are both expressed in growth plate chondrocytes. Although the effect of estrogen on bone growth is well known, the mechanism of action on longitudinal bone growth is poorly understood. We first studied longitudinal bone growth in female mice lacking ERalpha, ERbeta or both receptors (Paper I). Analysis of bone length and growth plate morphology revealed that ERbeta inhibits longitudinal bone growth. Moreover, in the presence of high serum concentrations of estrogen, stimulation of ERbeta induces growth plate fusion. Expression of both androgen and estrogen receptors were demonstrated within the growth plate. To study any co-interaction between estrogen and androgen receptors, we treated female ovariectomized rats with either estrogen, dihydrotestosterone (DHT) or the combination (Paper II). We found that estrogen inhibits longitudinal bone growth and growth plate height whereas DHT is able to counteract this effect. The effect is likely to be associated with systemic actions of these hormones since we found that decreased IGF-I levels in estrogen treated rats were restored by DHT. Estrogen regulates longitudinal bone growth either indirectly, via the modulation of the GH/IGF-I axis, or directly, via binding to estrogen receptors. Direct effects of estrogen were studied in a human chondrocytic cell line and in cultured fetal rat metatarsal bones (Paper III). We show that chondrocytes can synthesize estrogen by themselves and thereby mimic the effect of exogenous estrogen treatment. We also show that locally produced estrogen stimulates chondrocyte proliferation, inhibits apoptosis and maintains bone growth. Selective estrogen receptor modulators (SERMs) are promising drugs to modulate longitudinal growth offering fewer side-effects. We found that tamoxifen, a well known SERM, diminishes bone growth potential in cultured fetal rat metatarsal bones through selective apoptotic elimination of stem-like chondrocytes (Paper IV). Tamoxifen triggers the Fas/FasL apoptotic pathway and activates the caspases-8, -9 and 3. This thesis increases our understanding of how estrogen exerts its effect on longitudinal bone growth. This knowledge may be valuable for the development of new treatment strategies in children with growth disturbances, e.g. using SERMs to modulate the timing of growth plate fusion and thereby inhibit or stimulate the remaining growth potential

Activation of mTORC1 in chondrocytes does not affect proliferation or differentiation, but causes the resting zone of the growth plate to become disordered

There are several pitfalls associated with research based on transgenic mice. Here, we describe our interpretation and analysis of mTORC1 activation in growth plate chondrocytes and compare these to a recent publication (Yan et al., Nature Communications 2016, 7:11151). Both laboratories employed TSC1-floxed mice crossed with collagen type 2-driven Cre (Col2-Cre), but drew substantially different conclusions. It was reported that activation of mechanistic target of rapamycin complex 1 (mTORC1) via Tsc1 ablation promotes the hypertrophy of growth plate chondrocytes, whereas we observe only disorganization in the resting zone, with no effect on chondrocyte hypertrophy or proliferation. Here, we present our data and discuss the differences in comparison to the earlier phenotypic characterization of TSC1 ablation in cartilage. Importantly, we detect Col2-Cre activity in non-cartilaginous tissues (including the brain) and discuss it in relation to other studies reporting non-cartilaginous expression of collagen alpha(1) II. Altogether, we conclude that mouse phenotypes following genetic ablation using Col2-Cre should be interpreted with care. We also conclude that activation of mTORC1 by TSC1 ablation in postnatal chondrocytes with inducible Col2-Cre (Col2-CreERt) leads to disorganization of the resting zone but causes no changes in chondrocyte proliferation or differentiation. Keywords: Chondrocyte, Growth plate, mTORC1, Tsc1, Knockout mice, Col2-Cre, Cr

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

<p>Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H⁺-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A₁ or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.</p

Parathyroid hormone/parathyroid hormone-related protein receptor signaling is required for maintenance of the growth plate in postnatal life

Parathyroid hormone (PTH)-related protein (PTHrP), regulated by Indian hedgehog and acting through the PTH/PTHrP receptor (PPR), is crucial for normal cartilage development. These observations suggest a possible role of PPR signaling in the postnatal growth plate; however, the role of PPR signaling in postnatal chondrocytes is unknown. In this study, we have generated tamoxifen-inducible and cartilage-specific PPR KO mice to evaluate the physiological role of PPR signaling in postnatal chondrocytes. We found that inactivation of the PPR in chondrocytes postnatally leads to accelerated differentiation of chondrocytes, followed by disappearance of the growth plate. We also observed an increase of TUNEL-positive cells and activities of caspase-3 and caspase-9 in the growth plate, along with a decrease in phosphorylation of Bad at Ser155 in postnatal PPR KO mice. Administration of a low-phosphate diet, which prevents apoptosis of chondrocytes, prevented the disappearance of the growth plate. Taken together, these observations suggest that the major consequences of PPR activation are similar in both the fetal and postnatal growth plates. Moreover, chondrocyte apoptosis through the activation of a mitochondrial pathway may be involved in the process of premature disappearance of the growth plate by postnatal inactivation of the PPR in chondrocytes

Repair of Damaged Articular Cartilage: Current Approaches and Future Directions

Articular hyaline cartilage is extensively hydrated, but it is neither innervated nor vascularized, and its low cell density allows only extremely limited self-renewal. Most clinical and research efforts currently focus on the restoration of cartilage damaged in connection with osteoarthritis or trauma. Here, we discuss current clinical approaches for repairing cartilage, as well as research approaches which are currently developing, and those under translation into clinical practice. We also describe potential future directions in this area, including tissue engineering based on scaffolding and/or stem cells as well as a combination of gene and cell therapy. Particular focus is placed on cell-based approaches and the potential of recently characterized chondro-progenitors; progress with induced pluripotent stem cells is also discussed. In this context, we also consider the ability of different types of stem cell to restore hyaline cartilage and the importance of mimicking the environment in vivo during cell expansion and differentiation into mature chondrocytes