Zhongyong Li

Functional gap junctions between osteocytic and osteoblastic cells.

Morphological evidence shows that osteocytes, bone cells that exist enclosed within bone matrix, are connected to one another and to surface osteoblasts via gap junctions; however, it is unknown whether these gap junctions are functional. Using a newly established murine osteocytic cell line MLO‐Y4, we have examined functional gap junctional intercellular communication (GJIC) between osteocytic cells and between osteocytic and osteoblastic cells. In our hands, MLO‐Y4 cells express phenotypic characteristics of osteocytic cells including a stellate morphology, low alkaline phosphatase activity, and increased osteocalcin messenger RNA (mRNA) compared with osteoblastic cells. Northern and Western blot analysis revealed that MLO‐Y4 cells express abundant connexin 43 (Cx43) mRNA and protein, respectively. Lucifer yellow dye transferred from injected to adjacent cells suggesting that osteocytic cells were functionally coupled via gap junctions. Functional GJIC between osteocytic and osteoblastic (MC3T3‐E1) cells was determined by monitoring the passage of calcein dye between the two cell types using a double labeling technique. The ability of bone cells to communicate a mechanical signal was assessed by mechanically deforming the cell membrane of single MLO‐Y4 cells, cocultured with MC3T3‐E1 cells. Deformation induced calcium signals in MLO‐Y4 cells and those elicited in neighboring MC3T3‐E1 cells were monitored with the calcium sensitive dye Fura‐2. Our results suggest that osteocytic MLO‐Y4 cells express functional gap junctions most likely composed of Cx43. Furthermore, osteocytic and osteoblastic cells are functionally coupled to one another via gap junctions as shown by the ability of calcein to pass between cells and the ability of cells to communicate a mechanically induced calcium response. (J Bone Miner Res 2000;15:209–217)

Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells

Osteocytes, the predominant cells in bone, are postulated to be responsible for sensing mechanical and electrical stimuli, transducing signals via gap junctions. Osteocytes respond to induced shear by increasing connexin 43 (Cx43) levels, suggesting that they might be sensitive to physical stimuli like low‐frequency electromagnetic fields (EMF). Immature osteoblasts exhibit decreased intercellular communication in response to EMF but no change in Cx43. Here, we examined long term effects of pulsed EMF (PEMF) on MLO‐Y4 osteocyte‐like cells and ROS 17/2.8 osteoblast‐like cells. In MLO‐Y4 cell cultures, PEMF for 8 h/day for one, two or four days increased alkaline phosphatase activity but had no effect on cell number or osteocalcin. Transforming growth factor beta‐1 (TGF‐β1) and prostaglandin E2 were increased, and NO2‐ was altered. PEMFs effect on TGF‐β1 was via a prostaglandin‐dependent mechanism involving Cox‐1 but not Cox‐2. In ROS 17/2.8 cells, PEMF for 24, 48 or 72 h did not affect cell number, osteocalcin mRNA or osteocalcin protein. PEMF reduced Cx43 protein in both cells. Longer exposures decreased Cx43 mRNA. This indicates that cells in the osteoblast lineage, including well‐differentiated osteoblast‐like ROS 17/2.8 cells and terminally differentiated osteocyte‐like MLO‐Y4 cells, respond to PEMF with changes in local factor production and reduced Cx43, suggesting decreased gap junctional signaling.

Inhibiting gap junctional intercellular communication alters expression of differentiation markers in osteoblastic cells.

Gap junctional intercellular communication (GJIC) may contribute to cellular differentiation. To examine this possibility in bone cells we examined markers of cellular differentiation, including alkaline phosphatase, osteocalcin, and osteopontin, in ROS17/2.8 cells (ROS), a rat osteoblastic cell line expressing phenotypic characteristics of fully differentiated osteoblasts. We utilized ROS rendered communication deficient either by stable transfection with antisense cDNA to connexin 43 (Cx43), the predominant gap junction protein in bone (RCx16 cells), or by overexpression of Cx45, a gap junction protein not normally expressed in ROS (ROS/Cx45 cells). Both RCx16 and ROS/Cx45 cells displayed reduced dye coupling and Cx43 protein expression relative to ROS, control transfectants, and ROS/Cx45tr, ROS cells expressing carboxylterminal truncated Cx45. Steady-state mRNA levels for osteocalcin as well as alkaline phosphatase activity, two markers of osteoblastic differentiation, were also reduced in poorly coupled RCx16 and ROS/Cx45 cells. On the other hand, steady-state mRNA levels for osteopontin increased slightly in RCx16 and ROS/Cx45 cells. These results suggest that GJIC at least partly contributes to the regulation of expression of markers of osteoblastic differentiation.

Expression of Connexin 43 in Rat Mandibular Bone and Periodontal Ligament (PDL) Cells during Experimental Tooth Movement

Bone remodeling in response to force requires the coordinated action of osteoblasts, osteoclasts, osteocytes, and periodontal ligament cells. Coordination among these cells may be mediated, in part, by cell-to-cell communication via gap junctions. This study tests the hypothesis that the regulation of expression of connexin 43, a gap junction protein, is part of the transduction mechanism between force as applied to bone during orthodontic tooth movement and bone remodeling. To test this hypothesis, we examined connexin 43 expression in a rat model system of experimental tooth movement. To establish the model, we extracted maxillary first molars to initiate supra-eruption of opposing mandibular molars. The rats were killed at 0, 6, 12, 24, and 48 hrs post-extraction. The mandibles were removed, demineralized, and embedded in paraffin. To localize connexin 43 protein and mRNA, we used a specific antibody for immunohistochemistry and a specific cDNA probe for in situ hybridization. Western and Northern blot analyses were used to assess the specificity of the connexin 43 antibody and cDNA probe, respectively. We found connexin 43 protein expressed by osteoclasts (++++) and periodontal ligament cells (+++) in compression zones, and by osteoblasts (++++) and osteocytes (++++) in tension zones of the periodontal ligament. In addition, connexin 43 mRNA was found in some bone and periodontal ligament cells. Connexin 43 protein was found, by densitometric analysis, to be higher in the periodontal ligament after exposure to force compared with controls (P < 0.001). The number of osteocytes expressing connexin 43 48 hrs after molar extraction was also significantly greater in bone subjected to tension when compared with controls (P < 0.001). The results of this study support the hypothesis that connexin 43 plays a role in the coordination of events during experimentally induced alveolar bone remodeling.

Fluid flow-induced prostaglandin E2 response of osteoblastic ROS 17/2.8 cells is gap junction-mediated and independent of cytosolic calcium

It has been well demonstrated that bone adapts to mechanical loading. To accomplish this at the cellular level, bone cells must be responsive to mechanical loading (mechanoresponsive). This can occur via such mechanisms as direct cell deformation or signal transduction via complex pathways involving chemotransport, hormone response, and/or gene expression, to name a few. Mechanotransduction is the process by which a bone cell senses a biophysical signal and elicits a response. While it has been demonstrated that bone cells can respond to a wide variety of biophysical signals including fluid flow, stretch, and magnetic fields, the exact pathways and mechanisms involved are not clearly understood. We postulated that gap junctions may play an important role in bone cell responsiveness. Gap junctions (GJ) are membrane-spanning channels that physically link cells and support the transport of small molecules and ions in the process of gap junctional intercellular communication (GJIC). In this study we examined the role of GJ and GJIC in mechanically stimulated osteoblastic cells. Following fluid flow stimulation, we quantified prostaglandin E(2) (PGE(2)) (oscillatory flow) and cytosolic calcium (Ca(2+)) (oscillatory and steady flow) responses in ROS 17/2.8 cells and a derivative of these cells expressing antisense cDNA for the gap junction protein connexin 43 (RCx16) possessing significantly different levels of GJIC. We found that the ROS17/2.8 cells possessing increased GJIC also exhibited increased PGE(2) release to the supernatant following oscillatory fluid flow stimulation in comparison to coupling-decreased RCx16 cells. Interestingly, we found that neither osteoblastic cell line responded to oscillatory or steady fluid flow stimulation with an increase in Ca(2+). Thus, our results suggest that GJ and GJIC may be important in the mechanotransduction mechanisms by which PGE(2) is mechanically induced in osteoblastic cells independent of Ca(2+).

Breast cancer metastatic potential: correlation with increased heterotypic gap junctional intercellular communication between breast cancer cells and osteoblastic cells.

The breast cancer metastasis‐suppressor gene BRMS1 is downregulated in metastatic breast cancer cells. Previous reports have shown restoration of gap junctional intercellular communication (GJIC) in the metastatic human breast carcinoma cell line MDA‐MB‐435 (435) transfected with BRMS1 cDNA. Metastasis, to a large extent in most breast cancers, occurs to bone. However, the reason for this preferential metastasis is not known. We explored cell‐to‐cell communication between 435 carcinoma cells and a human osteoblastic cell line, hFOB1.19, to determine whether carcinoma cells can form gap junctions with bone cells and to explore the role of these heterotypic gap junctions and the BRMS1 gene in breast cancer metastasis to bone. 435 cells displayed greater cell‐to‐cell communication with hFOB 1.19 cells than with themselves. Transfection of BRMS1 into 435 cells increased homotypic gap junctional communication but did not significantly affect heterotypic communication with hFOBs. However, heterotypic communication of BRMS1 transfectants with hFOB cells was reduced relative to homotypic communication. In contrast, parental 435 cells displayed greater heterotypic communication with hFOBs relative to homotypic communication. Our results suggest that there are differences in the relative homotypic and heterotypic GJIC of metastasis‐capable and ‐suppressed cell lines.

Expressing connexin 43 in breast cancer cells reduces their metastasis to lungs

Recently the concept that gap junctions play a role in cancer cell metastasis has emerged. However, the mechanism by which this might occur is unknown. To examine this issue a metastatic breast cancer cell line, MDA-MB-435, was stably transfected with human Cx43 cDNA. Four clones of 435 transfectants (435/Cx43+ c1, c6, c8, c14) and two clones of plasmid control (435/hy) were isolated and examined in this study. We found that expressing Cx43 in MDA-MB-435 cells decreased their expression of Cx32 but did not affect gap junctional intercellular communication, migration or invasion through Matrigel®. However, forced expression of Cx43 decreased the growth of MDA-MB-435 cells, decreased expression of N-cadherin, which is frequently associated with an aggressive phenotype, and increased MDA-MB-435 sensitivity to apoptosis. More importantly, there were fewer lung metastases in mice injected with 435/Cx43+ cells relative to mice injected with 435/hy. These results suggest that expressing Cx43 in breast cancer cells decreases their metastatic potential through a mechanism independent of gap junctional communication but, rather, related to N-cadherin expression and apoptosis.

Alterations in Cx43 and OB-cadherin affect breast cancer cell metastatic potential

Emerging evidence suggests that gap junctional intercellular communication (GJIC) and expression of connexins (Cx) contribute to the metastatic potential of breast cancer cells. To more directly address this, an aggressive bone metastasis breast cancer cell line, MDA-MET (MET), was stably transfected with human Cx43 cDNA (MET/Cx43+). Focusing on clone 28 of MET/Cx43+, we demonstrated that GJIC, Cx43 protein and Cx43 mRNA were significantly increased in MET/Cx43+ cells relative to MET, the plasmid control for the Cx43 transfectants (MET/HY) and a metastatic breast cancer cell that is less metastatic to bone than MET, MDA-MB-231. Cx26 mRNA was also increased in MET/Cx43+ clone 28 cells while mRNA for Cx32, Cx37, Cx40 and Cx45 were not detected in any of the breast cancer cell lines examined. MET/Cx43+ clone 28 invasiveness was decreased by 33% relative to MET/HY, while their ability to migrate was unchanged. The ability of MET/Cx43+ clone 28 cells to adhere to hFOB and HUV-EC-C cells was decreased approximately 30% and 70%, respectively, relative to MET and MET/HY. E-cadherin and N-cadherin proteins were not detected in MET, MDA-MB-231, MET/Cx43+ clone 28 and MET/HY cells. However, OB-cadherin protein levels were decreased approximately 43% in MET/Cx43+ clone 28 relative to MET/HY cells. These findings suggest that GJIC and Cx43 expression contribute to breast cancer cell adhesion and migration, possibly through a mechanism involving OB-cadherin, and these changes in turn regulate the metastatic potential of breast cancer cells, especially to bone.

Age‐related changes in gap junctional intercellular communication in osteoblastic cells

Aging demonstrates deleterious effects upon the skeleton which can predispose an individual to osteoporosis and related fractures. Despite the well‐documented evidence that aging decreases bone formation, there remains little understanding whereby cellular aging alters skeletal homeostasis. We, and others, have previously demonstrated that gap junctions—membrane‐spanning channels that allow direct cell‐to‐cell conductance of small signaling molecules—are critically involved in osteoblast differentiation and skeletal homeostasis. We examined whether the capacity of rat osteoblastic cells to form gap junctions and respond to known modulators of gap junction intercellular communication (GJIC) was dependent on the age of the animal from which they were isolated. We observed no effect of age upon osteoblastic Cx43 mRNA, protein or GJIC. We also examined age‐related changes in PTH‐stimulated GJIC. PTH demonstrated age‐dependent effects upon GJIC: Osteoblastic cells from young rats increased GJIC in response to PTH, whereas there was no change in GJIC in response to PTH in osteoblastic cells from mature or old rats. PTH‐stimulated GJIC occurred independently of changes in Cx43 mRNA or protein expression. Cholera toxin significantly increased GJIC in osteoblastic cells from young rats compared to those from mature and old rats. These data demonstrate an age‐related impairment in the capacity of osteoblastic cells to generate functional gap junctions in response to PTH, and suggest that an age‐related defect in G protein‐coupled adenylate cyclase activity at least partially contributes to decreased PTH‐stimulated GJIC.

Connexin 43 gene expression in mice with cardiopulmonary developmental defects

Gap junctions are vital for cellular integrity, including homeostasis, morphogenesis, differentiation and growth in normal development of organs such as heart. Connexin 43 (Cx43) is a major gap junction protein. Our cDNA microarray analysis of normal and nitrofen-exposed neonatal mice with hypoplastic lungs, associated congenital diaphragmatic hernia (CDH) and heart developmental defects showed up-regulation of Cx43. Our objective was to establish if cardiopulmonary defects in nitrofen-exposed mice may be linked to altered expression of the Cx43 gene. We addressed our objective by performing northern blot analysis, real-time RT-PCR, immunoblotting and immunohistochemistry by localizing Cx43 in hearts and lungs of normal and nitrofen-exposed mice at different gestational stages. The data confirmed up-regulation of Cx43 expression in both hearts and lungs of CDH neonate mice and in lungs at other developmental stages except the pseudoglandular stage. However, Cx43 protein levels were either the same or less in hearts and lungs of nitrofen-exposed mice than in normal tissues except in pseudoglandular lungs. Different expressions of mRNA and protein suggest possible post-transcriptional or translational defects in Cx43. We observed dysmorphic hearts with exaggerated interventricular grooves and deep notches at the apex of the hearts in nitrofen-exposed fetal/neonatal mice; narrowed pulmonary out-flow and various degrees of craniofacial defects in 15-20% of the affected mice. Our data suggest a possible involvement of Cx43 in craniofacial, heart and lung defects in nitrofen-exposed mice. Such cardiopulmonary defects are also observed in human newborns with CDH. Thus, the murine data may help elucidate the pathways of cardiopulmonary defects in the human newborn condition.