The osteocyte lineage

The osteocyte resides in the lacuna/canalicular system in bone and has been hypothesized to orchestrate local bone remodeling. Certainly the identification of the osteocyte as the source of Sclerostin, a molecule that regulates osteoblast function, has supported this possibility. As our understanding of this cell increases it has become clear that it has more far reaching influence than simply local bone turnover activity. The osteocyte is also the source of DMP-1 and FGF-23, the later being a hormone that regulates kidney function in terms of phosphate uptake. We now see the osteocyte as having important roles both locally in the skeleton and also in other distant tissues. The study of osteocyte biology has reached a particularly exiting level of maturity and illustrates the value of this cell type as a drug discovery target.     

The response of bone to mechanical loading and disuse: fundamental principles and influences on osteoblast/osteocyte homeostasis

Bone’s response to increased or reduced loading/disuse is a feature of many clinical circumstances, and our daily life, as habitual activities change. However, there are several misconceptions regarding what constitutes loading or disuse and why the skeleton gains or loses bone. The main purpose of this article is to discuss the fundamentals of the need for bone to experience the effects of loading and disuse, why bone loss due to disuse occurs, and how it is the target of skeletal physiology which drives pathological bone loss in conditions that may not be seen as being primarily due to disuse. Fundamentally, if we accept that hypertrophy of bone in response to increased loading is a desirable occurrence, then disuse is not a pathological process, but simply the corollary of adaptation to increased loads. If adaptive processes occur to increase bone mass in response to increased load, then the loss of bone in disuse is the only way that adaptation can fully tune the skeleton to prevailing functional demands when loading is reduced. The mechanisms by which loading and disuse cause bone formation or resorption are the same, although the direction of any changes is different. The osteocyte and osteoblast are the key cells involved in sensing and communicating the need for changes in mass or architecture as a result of changes in experienced loading. However, as those cells are affected by numerous other influences, the responses of bone to loading or disuse are not simple, and alter under different circumstances. Understanding the principles of disuse and loading and the mechanisms underlying them therefore represents an important feature of bone physiology and the search for targets for anabolic therapies for skeletal pathology.     

Cell-to-cell interactions in bone

Bone development (modeling) occurs by migration, aggregation, and condensation of immature osteo/chondroprogenitor cells to form the cartilaginous anlage. This process requires precisely controlled cell-cell interactions. Likewise, bone remodeling in the adult skeleton is a dynamic process that requires coordinated cellular activities among osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. The cooperative nature of both bone modeling and remodeling requires tightly regulated mechanisms of intercellular recognition and communication that permit the cells to sort and migrate, synchronize activity, equalize hormonal responses, and diffuse locally generated signals. Osteoblasts and osteocytes achieve these interactions through cadherin-based adherens junctions as well as by formation of communicating junctions, gap junctions. This review examines the current knowledge of how direct cell-to-cell interactions modulate osteoblast function.     

Protein phosphatase modulation of the intercellular junctional communication: importance in cardiac myocytes

The rhythmic contraction of a four-chambered heart is a highly co-ordinated process, requiring the sequential activation of pacemaker cells and the propagation of activity throughout the whole myocardium. Gap-junctional channels, providing enclosed conduits for direct cell-to-cell transfer of ions and small molecules between adjacent cells, allow depolarising currents to flow from excited to non-excited regions of the network and a gradual spreading of the action potential. Gap-junctional channels are dodecamers of transmembrane proteins belonging in chordates to the connexin (Cx) family. In mammalian hearts, cardiomyocytes most prominently express junctional channels built of three Cxs: Cx40, Cx43 and Cx45. As with the great majority of Cx, they are phosphoproteins and exist under different phosphorylated levels. Phosphorylation, a widespread post-translational modification of proteins, is a primary means of mediating signal transduction events that control numerous cellular processes via a highly regulated dynamic interplay of protein kinases (PKs) and protein phosphatases (PPs). These processes appear implicated in the regulation of gap-junctional communication at several stages of the Cx lifecycle, including intracellular Cx trafficking, connexon assembly and disassembly, Cx degradation as well as the gating of gap-junction channels, but the underlying mechanisms remain poorly understood. Although PKs have an established role in this process, less is known about the involvement of PPs. The present review examines the roles played by protein dephosphorylation catalysers in the regulation of the gap-junctional communication in general, with a special focus on the junctional communication between cardiac cells.     

Gap junctional regulation of signal transduction in bone cells

The role of gap junctions, particularly that of connexin43 (Cx43), has become an area of increasing interest in bone physiology. An abundance of studies have shown that Cx43 influences the function of osteoblasts and osteocytes, which ultimately impacts bone mass acquisition and skeletal homeostasis. However, the molecular details underlying how Cx43 regulates bone are only coming into focus and have proven to be more complex than originally thought. In this review, we focus on the diverse molecular mechanisms by which Cx43 gap junctions and hemichannels regulate cell signaling pathways, gene expression, mechanotransduction and cell survival in bone cells. This review will highlight key signaling factors that have been identified as downstream effectors of Cx43 and the impact of these pathways on distinct osteoblast and osteocyte functions.     

Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication

Gap junction channels are made of a family proteins called connexins. The best-studied type of connexin, Connexin43 (Cx43), is phosphorylated at several sites in its C-terminus. The tumor-promoting phorbol ester TPA strongly inhibits Cx43 gap junction channels. In this study we have investigated mechanisms involved in TPA-induced phosphorylation of Cx43 and inhibition of gap junction channels. The data show that TPA-induced inhibition of gap junction intercellular communication (GJIC) is dependent on both PKC and the MAP kinase pathway. The data suggest that PKC-induced activation of MAP kinase partly involves Src-independent trans-activation of the EGF receptor, and that TPA-induced shift in SDS-PAGE gel mobility of Cx43 is caused by MAP kinase phosphorylation, whereas phosphorylation of S368 by PKC does not alter gel migration of Cx43. We also show that TPA, in addition to phosphorylation of S368, also induces phosphorylation of S255 and S262, in a MAP kinase-dependent manner. The data add to our understanding of the molecular mechanisms involved in the interplay between signaling pathways in regulation of GJIC.     

Nontransformed cells can normalize gap junctional communication with transformed cells

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.     

Orientation of collagen at the osteocyte lacunae in human secondary osteons

This work characterizes an aspect of human bone micro-structure, pertinent to fracture initiation and arrest. It addresses how the orientation of elementary components proximate to osteocyte lacunae influences secondary osteon micro-biomechanics. New data at the perilacunar region concerning orientation of collagen-apatite, and prior data on collagen orientation outside the perilacunar region, are incorporated in a novel simulation of osteons to investigate how orientation relates to strains and stresses during mechanical testing. The perilacunar region was observed by confocal microscopy within single lamellar specimens, isolated from osteons. The specimens were separated by extinct or bright appearance in transverse section under circularly polarizing light. This is because synchrotron diffraction and confocal microscopy had established that each type, away from the perilacunar region, corresponds to specific dominant collagen orientation (extinct lamellae's dominant collagen forming small angles with the original osteon axis, while the bright lamellae's forms larger angles). Morphometry of serial confocal images of each perilacunar region showed collagen orientation generally following the orientation of canaliculi, circumambiently-perpendicular to the lacuna. The lacunae tilted relative to the lamellar walls were more numerous in extinct than in bright lamella. Their apices were less likely in extinct than bright lamella to show collagen following the canalicular orientation. The simulation of osteocyte lacunae in osteons, under tension or compression loading, supports the hypothesis that collagen orientation affects strains and stresses at the equatorial perilacunar region in conjunction with the presence of the lacuna. We further conjecture that collagen orientation diverts propagation of micro-cracks initiating from apices.     

Passage through vertebrate gap junctions of 17/18 kDa molecules is primarily dependent upon molecular configuration

In fish, amphibians and mammals, gap junctions of some cells allow passage of elongate molecules as large as 18 kDa, while excluding smaller, less elongate molecules. Fluorescently labeled Calmodulin (17 kDa) and fluorescently labeled Troponin-C (18 kDa), when microinjected into oocytes of Danio rerio, Xenopus laevis or Mus domestica, were able to transit the gap junctions between these oocytes and the granulosa cells which surrounded them. Co-microinjected with these Ca²⁺-binding proteins, Texas-red-labeled dextran (10 kDa) remained in the microinjected cell. Osteocalcin (6 kDa), also a Ca²⁺-binding protein, but with a wide “V” shape proved unable to transit these gap junctions. Calmodulin, but not Troponin-C, was able to transit gap junctions of gonadotropin treated WB cells in culture. We show evidence that molecules as large as 18 kDa can pass through some vertebrate gap junctions, both homologous and heterologous, and that it is primarily molecular configuration which governs gap junctional permeability.     

Expression and function of Dlx genes in the osteoblast lineage

Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5^−/−/Dlx6^−/− mice have more severe craniofacial and limb defects than Dlx5^−/−, some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5^−/− mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature osteoblasts, whereas Dlx3 is very highly expressed in differentiated osteoblasts and osteocytes. In situ hybridization and Northern blot analysis demonstrated the presence of endogenous Dlx3 mRNA within osteoblasts and osteocytes. Dlx3 strongly upregulates osteoblastic markers with a potency comparable to Dlx5. Cloned chick or mouse Dlx6 showed stimulatory effects on osteoblast differentiation. Our results suggest that Dlx2 and Dlx6 have the potential to stimulate osteoblastic differentiation and may compensate for the absence of Dlx5 to produce relatively normal osteoblastic differentiation in Dlx5 knockout mice, while Dlx3 may play a distinct role in late stage osteoblast differentiation and osteocyte function.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

The cataract causing Cx50-S50P mutant inhibits Cx43 and intercellular communication in the lens epithelium

Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function. Using dual whole-cell voltage clamp, immunofluorescent microscopy and in situ dye transfer analysis we identified a unique interaction between the mutant subunit and wild-type Cx43. In paired Xenopus oocytes, co-expression of Cx50-S50P with Cx43 reduced electrical coupling ≥ 90%, without a reduction in protein expression. In transfected cells, Cx50-S50P did not target to cell-cell interfaces by itself, but co-expression of Cx50-S50P with Cx43 resulted in its localization at areas of cell-cell contact. We used Cx43 conditional knockout, Cx50 knockout and Cx50-S50P mutant mice to examine this interaction in vivo. Mice expressing both Cx43 and Cx50-S50P in the lens epithelium revealed a unique expression pattern for Cx43 and a reduction in Cx43 protein. In situ dye transfer experiments showed that the Cx50-S50P mutant, but not the Cx50, or Cx43 conditional knockout, greatly inhibited epithelial cell gap junctional communication in a manner similar to a double knockout of Cx43 and Cx50. The inhibitory affects of Cx50-S50P lead to diminished electrical coupling in vitro, as well as a discernable reduction in epithelial cell dye permeation. These data suggest that dominant inhibition of Cx43 mediated epithelial cell coupling may play a role in the lens pathophysiology caused by the Cx50-S50P mutation.     

Expression and function of connexins in the epidermis, analyzed with transgenic mouse mutants

Eight different connexins are expressed in mouse epidermis with overlapping expression patterns in different epidermal layers. Analyses of mice with deficiency or modifications of distinct connexins yielded insights into the large variety of connexins in the epidermis. Connexin43 (Cx43) deficiency in mouse epidermis resulted in a significant acceleration of wound closure. Truncation by 125 amino acid residues of the Cx43 C-terminal region led to an altered epidermal expression pattern of Cx43 and defective development of the epidermal water barrier in transgenic mice, although the truncated Cx43 protein could still form open gap junctional channels in transfected HeLa cells. Thus, the phenotypic abnormalities observed in mice with truncated Cx43 protein (Cx43K258Stop) are more likely due to defective regulation of this protein rather than the closed Cx43 channel. Our studies of connexin-deficient mice revealed an extensive redundancy of connexins expressed in mouse epidermis. Epidermal connexins seem to form two functional groups in which deficiency of one connexin isoform can be compensated by other connexin isoforms of the same group.     

Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice

The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.     

NBT-II carcinoma behaviour is not dependent on cell-cell communication through gap junctions

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.