Boning up on glypicans—opportunities for new insights into bone biology

Bone formation is remarkable for the convergence in the activity of four major signalling pathways, the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH) and wingless‐integrated (WNT) pathways. These pathways cooperate in morphogenetic, proliferative and differentiative processes that underpin the development, growth and repair of skeletal structures. They are regulated by pathway‐specific modulators and by another class of molecules, the glypicans. Glypicans are proteoglycans located on the cell surface, where they act as coreceptors to promote or inhibit signalling by ligands of the BMP, FGF, HH and WNT pathways, through protein–protein and protein–carbohydrate interactions. In this review, we discuss glypican structure, expression and function in the context of bone development and growth, with emphasis on the long bone growth plate where five of the six glypicans are expressed in overlapping patterns in the chondrogenic zone. Analyses of gene knockout models and the human conditions of Simpson–Golabi–Behmel syndrome and omodysplasia, which arise from mutations in glypican 3 (GPC3) and GPC6, respectively, highlight both subtle and striking effects of glypicans on bone growth. We draw attention to challenges and areas of opportunity, where the actions of glypicans on BMP, FGF, HH and WNT signalling might be profitably studied to help illuminate the complex interplay of signalling that drives bone growth. Copyright © 2013 John Wiley & Sons, Ltd.     

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20-30% upon mechanical loading within 30min. We showed that the RGD-integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca(2+) channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.     

Visual selection: Neurons that make up their minds.

Fresh evidence about how cells in frontal cortex make decisions has come from experiments in which information is partly withheld from them.     

Boning up on autophagy: The role of autophagy in skeletal biology

From an evolutionary perspective, the major function of bone is to provide stable sites for muscle attachment and affording protection of vital organs, especially the heart and lungs (ribs) and spinal cord (vertebrae and intervertebral discs). However, bone has a considerable number of other functions: serving as a store for mineral ions, providing a site for blood cell synthesis and participating in a complex system-wide endocrine system. Not surprisingly, bone and cartilage cell homeostasis is tightly controlled, as is the maintenance of tissue structure and mass. While a great deal of new information is accruing concerning skeletal cell homeostasis, one relatively new observation is that the cells of bone (osteoclasts osteoblasts and osteocytes) and cartilage (chondrocytes) exhibit autophagy. The focus of this review is to examine the significance of this process in terms of the functional demands of the skeleton in health and during growth and to provide evidence that dysregulation of the autophagic response is involved in the pathogenesis of diseases of bone (Paget disease of bone) and cartilage (osteoarthritis and the mucopolysaccharidoses). Delineation of molecular changes in the autophagic process is uncovering new approaches for the treatment of diseases that affect the axial and appendicular skeleton.     

Uncertainty in the niches that maintain haematopoietic stem cells

Haematopoietic stem cell (HSC) niches are specialized microenvironments that contain stem cells and regulate their maintenance. Cells at the interface of bone and the bone marrow (the endosteum) contribute to the creation of HSC niches. It remains uncertain whether this interface itself is a niche, or whether endosteal cells secrete factors that diffuse to nearby niches. Vascular and/or perivascular cells may also create niches as many HSCs are observed around sinusoidal blood vessels, and perivascular cells secrete factors that regulate HSC maintenance. Do endosteal and perivascular cells create distinct niches, or do they contribute to a common niche? We discuss a range of niche models consistent with recent evidence.     

Wolff's law and the problem of muscle attachment on resorptive surfaces of bone

During the growth of a bone, outer (periosteal) surfaces in many areas undergo normal remodeling processes involving resorptive removal. Attachments of muscles commonly occur on such outer resorptive surfaces. The cortex in these regions grows in an inward direction by bone deposition on endosteal surfaces. In some areas of a bone, a portion of a muscle can be inserted onto a depository surface, but other parts of the same muscle may be attached onto an adjacent resorptive surface. It has been generally assumed that the pull of a muscle acts to directly stimulate deposition of new bone, and that attachments of muscle are thereby responsible for determining the gross morphology of a whole bone. In view of the foregoing considerations, a re-evaluation and an expansion of this concept is now needed. Muscle pull, in many regions of a bone, can be associated with normal cortical recession (involving surface resorption) as well as with outward bone deposition.     

T-Box genes and developmental decisions that cells make

During gastrulation in vertebrate embryos, three definitive germ layers (ectoderm, mesoderm, and endoderm) are formed by organized and coordinated cell movements. In zebrafish, further subdivision of the mesoderm gives rise to the axial, adaxial and paraxial mesoderm. The axial mesoderm contributes to the prechordal plate and notochord whereas the adaxial and paraxial cells give rise to slow and fast muscles, respectively (Devoto et al., 1996; Blagden et al., 1997; Currie and Ingham, 1998). An inductive interaction in which the notochord plays an essential role will also provide an input in forming other specialized types of tissue contributing to the axial structures: the floor plate located dorsally to the notochord in the ventral spinal cord and the hypochord located ventrally of the notochord and deriving probably from the endoerm. It is known that despite the difference in developmental roles (Str?hle et al., 1993; Krauss et al., 1993), the floor plate and hypochord co-express a number of common molecular markers (Jan et al., 1995; our unpublished results) that may illustrate a certain similarity of their origin. Their close proximity to the notochord determines specialized features of these structures that differ substantially from the rest of the neural tube and endoderm, correspondingly. Once formed under the influence of the notochordal signaling, the floor plate will acquire an ability, similar to the notochord, to express genes of the Hedgehog family and several other groups of genes and to induce specification of ventral cell types in the neural tube during later development (for review, see Korzh, 1998). The biology of the hypochord is much less understood. It seems that the hypochord develops slightly later than the floor plate. It may be required for proper positioning of the dorsal aorta as well as induction of some other endoderm derivatives.     

Mechanical regulation of signaling pathways in bone

A wide range of cell types depend on mechanically induced signals to enable appropriate physiological responses. The skeleton is particularly dependent on mechanical information to guide the resident cell population towards adaptation, maintenance and repair. Research at the organ, tissue, cell and molecular levels has improved our understanding of how the skeleton can recognize the functional environment, and how these challenges are translated into cellular information that can site-specifically alter phenotype. This review first considers those cells within the skeleton that are responsive to mechanical signals, including osteoblasts, osteoclasts, osteocytes and osteoprogenitors. This is discussed in light of a range of experimental approaches that can vary parameters such as strain, fluid shear stress, and pressure. The identity of mechanoreceptor candidates is approached, with consideration of integrins, pericellular tethers, focal adhesions, ion channels, cadherins, connexins, and the plasma membrane including caveolar and non-caveolar lipid rafts and their influence on integral signaling protein interactions. Several mechanically regulated intracellular signaling cascades are detailed including activation of kinases (Akt, MAPK, FAK), β-catenin, GTPases, and calcium signaling events. While the interaction of bone cells with their mechanical environment is complex, an understanding of mechanical regulation of bone signaling is crucial to understanding bone physiology, the etiology of diseases such as osteoporosis, and to the development of interventions to improve bone strength.     

Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.     

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.     

Neural circuitry: seeing the parts that make the picture

What are the neural correlates of vision? A recent study on Drosophila has described the incredible neuronal diversity in the fly visual system, and traced the circuits that underlie color vision.     

Pre-receptorial regulation of steroid hormones in bone cells: insights on glucocorticoid-induced osteoporosis

In the past decades, concern on glucocorticoid-induced osteoporosis has increased with the widespread use of exogenous glucocorticoids (GC). Mature bone-forming cells (osteoblasts) are considered to be the principal site of action of GC in the skeleton. More likely, it is the entire cellular and molecular network surrounding these cells that is targeted by pharmacological doses of GC. Not only osteoblast and osteocyte metabolism, but the whole differentiation of mesenchymal stem cell toward the osteoblast lineage has been proven to be sensitive to GC. The effects of GC on this process are different according to the stage of differentiation of bone cell precursors. The presence of intact GC signalling is crucial for normal bone development and physiology, as opposed to the detrimental effect of high dose exposure. Both the physiological and pharmacological effects of GC are locally modulated by the activity of the 11β-hydroxysteroid dehydrogenase 1 (HSD1) that acts primarily as a glucocorticoid activator converting the inactive glucocorticoid (cortisone) into the active hormone (cortisol). We reviewed the metabolic and differentiation pathways controlled by GC signalling. These data have been merged with the recent evidences that 11β-HSD1 exert an important role by regulating the vulnerability of bone cells to GC. The different kinetics of 11β-HSD1 at various stage of differentiation and the GC-dependency of enzymatic activity have been presented.     

The shrew tamed by Wolff's law: Do functional constraints shape the skull through muscle and bone covariation?

Bone is a highly plastic tissue that reflects the many potential sources of variation in shape. Here, we focus on the functional aspects of bone remodeling. We choose the skull for our analyses because it is a highly integrated system that plays a fundamental role in feeding and is thus, likely under strong natural selection. Its principal mechanical components are the bones and muscles that jointly produce bite force and jaw motion. Understanding the covariations among these three components is of interest to understand the processes driving the evolution of the feeding apparatus. In this study, we quantitatively and qualitatively compare interactions between these three components in shrews from populations known to differ in shape and bite force. Bite force was measured in the field using a force transducer and skull shape was quantified using surface geometric morphometric approaches based on µCT‐scans of the skulls of same individuals. The masseter, temporalis, pterygoideus, and digastricus muscles of these individuals were dissected and their cross sectional areas determined. Our results show strong correlations between bite force and muscle cross sectional areas as well as between bite force and skull shape. Moreover, bite force explains an important amount of skull shape variation. We conclude that interactions between bone shape and muscle characteristics can produce different morpho‐functional patterns that may differ between populations and may provide a suitable target for selection to act upon. J. Morphol. 276:301–309, 2015. © 2014 Wiley Periodicals, Inc.     

Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi

Ectomycorrhizal fungi exhibit high diversity even in small monoculture forests. Roughly 20 to 35 species typically occupy such sites. Explanations for this diversity can be based on resource partitioning, disturbance, competition, or interaction with other organisms. Mycorrhizal fungi compete for two general classes of resources: host-derived carbon and soil or detritus derived mineral nutrients. Both types of resources are arrayed in space (e.g., soil depth, distance from tree) and time (e.g., season, host successional series). Some species seem to be partitioned in space and time at these scales, but the question of how widespread these patterns are remains largely unanswered. Mineral resources are distributed in discrete substrates in soil, litter, and within other soil microorganisms; the biochemical diversity exhibited by fungi may translate into differences in access to these resources among species. Small-scale natural disturbances that sever roots, mix soil horizons and litter layers, or change local pH and nutrient availability, are likely to create additional habitats for ectomycorrhizal fungi. Evidence from fruiting patterns and differences in colonization strategies suggest that such disturbances may be important for establishment of some species. Competitive replacement networks among species have the theoretical potential to increase diversity. The frequency of species replacements, observed co-infections of ectomycorrhizal fungi on single host roots, and high rates of rootlet turn-over all suggest that competition is important, but whether it plays a creative role in maintaining diversity remains to be demonstrated. Other organisms could be important in the maintenance of diversity, if they effect competition among mycorrhizal fungi. Bacteria and soil invertebrates are the most likely groups for such interactions. Technological advances in root observation and PCR methods for indentification of mycorrhizae make many of these theories testable.     

Lineage-negative bone marrow cells travel bidirectionally in the olfactory migratory stream but maintain hematopoietic phenotype

The mammalian olfactory system is a physiologically plastic region of the brain with the potential to support implanted stem cells. We performed direct injection of lineage-negative (lin-neg), green fluorescent protein-positive (GFP+) bone marrow cells into the olfactory bulb to assess cell survival and motility within the central nervous system (CNS). Before direct injection of 100,000 lin-neg cells, some of the C57/Bl mice received 1,000 cGy brain irradiation with the aim of disabling the endogenous reservoir of periventricular neural progenitor cells. Brain harvest took place up to 2 weeks after cell implantation. Brains were evaluated for presence of GFP positivity via fluorescence microscopy. Many GFP+ cells were identified within the turbinate neuroepithelium, olfactory bulb, and frontal lobe. Most of the cells that had traveled from the implantation site adopted an elongated, arborizing morphology consistent with cellular extensions arrayed in the direction of the rostral migratory stream (RMS). No difference was seen in brain-irradiated versus non-irradiated mice. Antibody staining revealed that these cells did not take on a neural, glial, or endothelial phenotype, while largely retaining their hematopoietic lineage as demonstrated by CD45 positivity.