Cross-talk between fibroblast growth factor and bone morphogenetic proteins regulates gap junction-mediated intercellular communication in lens cells

Homeostasis in the lens is dependent on an extensive network of cell-to-cell gap junctional channels. Gap junction-mediated intercellular coupling (GJIC) is higher in the equatorial region of the lens than at either pole, an asymmetry believed essential for lens transparency. Primary cultures of embryonic chick lens epithelial cells up-regulate GJIC in response to purified fibroblast growth factor (FGF)1/2 or to medium conditioned by vitreous bodies, the major reservoir of factors (including FGF) for the lens equator. We show that purified bone morphogenetic protein (BMP)2, -4, and -7 also up-regulate GJIC in these cultures. BMP2, -4, or both are present in vitreous body conditioned medium, and BMP4 and -7 are endogenously expressed by lens cells. Remarkably, lens-derived BMP signaling is required for up-regulation of GJIC by purified FGF, and sufficient for up-regulation by vitreous humor. This is the first demonstration of an obligatory interaction between FGF and BMPs in postplacode lens cells, and of a role for FGF/BMP cross-talk in regulating GJIC in any cell type. Our results support a model in which the angular gradient in GJIC in the lens, and thus proper lens function, is dependent on signaling between the FGF and BMP pathways.     

Modulation of gap junction-mediated intercellular communication in embryonic chick mesenchyme during tissue remodeling in vitro

Gap junction-mediated intercellular communication was analyzed in a model system in which tissue necrosis and remodeling could be modulated. This in vitro system, previously used for analysis of epithelial-mesenchymal tissue interaction, was modified to permit analysis of the presence and extent of intercellular communition by monitoring intercellular transfer of the micro-injected fluorescent dye, Lucifer Yellow. Light and transmission electronmicroscopy were employed to correlate the presence and degree of gap junctional communication (coupling) with tissue morphology. Digital image analysis was used to determine cell density and mitotic indices within the outgrowths of explants. Our results indicated that cell communication in outgrowths adjacent to necrotic foci within an explant was minimal or absent. Cell-coupling in outgrowths adjacent to a compartment of viable mesenchyme was significantly higher-equivalent to unseparated control cultures. A time-course study demonstrated correlation of increased levels of cell-coupling in outgrowths with the level of tissue remodeling within an explant. Our conclusions from these studies are that embryonic mesenchymal cell populations may be selectively uncoupled as a result of alterations in the microenvironment produced by a proximate impaired cell population. It is proposed that endogenous factors in the microenvironment (wound signals), emanating from impaired cell populations, regulate gap junction-mediated intercellular communication in adjacent viable tissue. Normal, unimpaired populations of cells surrounding an area of injury are thereby isolated from the effects of a potentially toxic environment. This could serve as a protective function in development and may represent, in a more general sense, part of the repertoire of events associated with tissue repair and remodeling.     

Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication.

We show that connexin expression and in vivo patterns of communication were dramatically altered in response to epidermal wounding. Six hours after injury, Cx26 was up-regulated in the differentiated cells proximal to the wound, but was down-regulated in cells located at the wound edge. In contrast, Cx31.1 and Cx43 were down-regulated in cells both peripheral to and at the wounded edge. These patterns of altered connexin expression were detectable as early as 2 h after wounding and were most pronounced in 24-h old wounds. Increased expression of Cx26 was still evident in the hyperproliferative epidermis of 6-day old wounds. In vivo dye transfer experiments with Lucifer yellow and neurobiotin confirmed that junctional communication patterns were altered in ways consistent with changes in connexin expression. The data thus suggest that intercellular communication is intimately involved in regulating epidermal wound repair.     

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

Introduction  

The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.     

BMP-2-modulated chondrogenic differentiation in vitro involves down-regulation of membrane-bound beta-catenin

Bone morphogenetic proteins (BMPs) are important regulators of cellular differentiation and embryonic development. Beta catenin mediated nuclear signaling has been implicated in BMP-2-modulated chondrogenic differentiation in the pluripotential stem cell line C3H10T1/2. However, there is little information on the functional role of beta catenin in BMP-2-modulated differentiation of primary nontransformed mesenchymal cells. Here, we present evidence to show that BMP-2-induced chondrogenic differentiation in high-density primary mesenchymal culture is associated with a significant decrease in membrane-bound beta catenin by 72 hours compared to controls. Nuclear localization of beta catenin is not detectable by immunofluorescence and the TCF-responsive reporter vector TOPFLASH shows only background activity during chondrogenic differentiation. BMP-2-treated cultures show reduced cell-cell adhesion by 72 hours, which correlates with the changes in levels of membrane-bound beta catenin. Up-regulation of membrane-bound beta catenin blocks the effect of BMP-2 on both chondrogenic differentiation and cell-cell adhesiveness. These findings suggest that BMP-2 can modulate the adhesivity of adherens junctions through regulation of membrane bound beta catenin.     

A relationship between gap junction-mediated intercellular communication and the in vitro developmental capacity of murine embryonic stem cells.

Two metabolic cooperation-deficient variants, 1P9 and 2P2[1], have been isolated from the embryonic stem cell line B2B2. Characterization of these cell lines has shown that both variants are severely restricted with respect to embryoid body differentiation capacity while a revertant isolated from 1P9, designated 2H4, has its differentiation phenotype restored to a level comparable with that of the parent line. These results are interpreted as indicating that the metabolic cooperation deficiency and the restricted differentiation phenotype of 1P9 are causally related. A revertant isolated from 2P2[1], designated H19, remains severely restricted with regard to embryoid body differentiation, suggesting that in these lines a secondary event deleterious to differentiation, but unrelated to metabolic cooperation, has taken place.     

Bone morphogenetic protein-2-induced transformation involves the activation of mammalian target of rapamycin

Bone morphogenetic protein-2 (BMP-2) is an evolutionary conserved protein that is essential for embryonic development. BMP-2 is highly expressed in approximately 98% of human lung carcinomas with little expression in normal lung tissues. BMP-2 has been shown to enhance mobility, invasiveness, and metastasis of cancer cell lines. During development, BMP-2 induces the proto-oncogene phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway to regulate stem cell differentiation. We show that BMP-2 induces the phosphorylation of mTOR in A549 and H1299 lung cancer cell lines, which is attenuated by the PI3K antagonists LY-294002 and wortmannin. p70S6 kinase, which is a direct downstream target of mTOR, is also regulated by BMP-2 in lung cancer cell lines. We find that BMP-2 induces cyclin E in A549 and H1299 cells, which is mediated by the PI3K/mTOR signaling pathway. The regulation of cyclin E by BMP-2 occurs through a Smad 1/5-independent mechanism. Forced expression of BMP-2 in A549 cells (A549/BMP-2) induces transformation as shown by an increase in foci formation. The mTOR antagonist, rapamycin, prevented foci formation of the A549/BMP-2 cells. This study provides evidence that BMP-2-mediated transformation of lung cancer cells involves the activation of the PI3K/mTOR signaling pathway.     

Gap junction-mediated intercellular communication in the immune system

Immune cells are usually considered non-attached blood cells, which would exclude the formation of gap junctions. This is a misconception since many immune cells express connexin 43 (Cx43) and other connexins and are often residing in tissue. The role of gap junctions is largely ignored by immunologists as is the immune system in the field of gap junction research. Here, the current knowledge of the distribution of connexins and the function of gap junctions in the immune system is discussed. Gap junctions appear to play many roles in antibody productions and specific immune responses and may be important in sensing danger in tissue by the immune system. Gap junctions not only transfer electrical and metabolical but also immunological information in the form of peptides for a process called cross-presentation. This is essential for proper immune responses to viruses and possibly tumours. Until now only 40 research papers on gap junctions in the immune system appeared and this will almost certainly expand with the increased mutual interest between the fields of immunology and gap junction research.     

Bone morphogenetic protein-2 stimulates Runx2 acetylation

Runx2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes. Runx2 is a major target of the bone morphogenetic protein (BMP) pathway. Genetic analysis has revealed that Runx2 is degraded through a Smurf-mediated ubiquitination pathway, and its activity is inhibited by HDAC4. Here, we demonstrate the molecular link between Smurf, HDACs and Runx2, in BMP signaling. BMP-2 signaling stimulates p300-mediated Runx2 acetylation, increasing transactivation activity and inhibiting Smurf1-mediated degradation of Runx2. HDAC4 and HDAC5 dea-cetylate Runx2, allowing the protein to undergo Smurf-mediated degradation. Inhibition of HDAC increases Runx2 acetylation, and potentiates BMP-2-stimulated osteoblast differentiation and increases bone formation. These results demonstrate that the level of Runx2 is controlled by a dynamic equilibrium of acetylation, deacetylation, and ubiquitination. These findings have important medical implications because BMPs and Runx2 are of tremendous interest with regard to the development of therapeutic agents against bone diseases.     

Bone marrow stroma inhibits proliferation and apoptosis in leukemic cells through gap junction-mediated cell communication

Normal and leukemic blood cell progenitors depend upon the bone marrow (BM) stroma with which they communicate through soluble and membrane-anchored mediators, adhesive interactions and gap junctions (GJ). Regarding hematopoiesis, it is believed that it can be influenced by connexin expression, but the exact role of GJ in cell death and proliferation is not clear. Using flow cytometry, we monitored the division rate of leukemic cell lines, communicating and not communicating with stromal cell line through GJ. We found that GJ-coupled cells (i) did not proliferate; (ii) were kept in G0; and (iii) were protected from drug-induced apoptosis when compared to either total or uncoupled cell population. We conclude that GJ coupling between stroma and leukemic lymphoblasts prevents proliferation, keeping cells in a quiescent state, thus increasing their resistance to antimitotic drugs. Since GJ are particularly abundant in the sub-endosteal environment, which harbors blood stem cells, we also asked which cells within the normal human BM communicate with the stroma. Using a primary BM stroma cell culture, our results show that 80% of CD34+ progenitors communicate through GJ. We propose that blood cell progenitors might be retained in the low-cycling state by GJ-mediated communication with the hematopoietic stroma.     

A novel role for FGF and extracellular signal-regulated kinase in gap junction-mediated intercellular communication in the lens.

Gap junction-mediated intercellular coupling is higher in the equatorial region of the lens than at either pole, a property believed to be essential for lens transparency. We show that fibroblast growth factor (FGF) upregulates gap junctional intercellular dye transfer in primary cultures of embryonic chick lens cells without detectably increasing either gap junction protein (connexin) synthesis or assembly. Insulin and insulin-like growth factor 1, as potent as FGF in inducing lens cell differentiation, had no effect on gap junctions. FGF induced sustained activation of extracellular signal-regulated kinase (ERK) in lens cells, an event necessary and sufficient to increase gap junctional coupling. We also identify vitreous humor as an in vivo source of an FGF-like intercellular communication-promoting activity and show that FGF-induced ERK activation in the intact lens is higher in the equatorial region than in polar and core fibers. These findings support a model in which regional differences in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function. Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.     

Bone morphogenetic protein-2 stimulates angiogenesis in developing tumors

Bone Morphogenetic Protein-2 (BMP-2) is highly overexpressed in the majority of patient-derived lung carcinomas. However, a mechanism revealing its role in cancer has not been established. Here we report that BMP-2 enhances the neovascularization of developing tumors. Recombinant BMP-2 stimulated blood vessel formation in tumors formed from A549 cells injected s.c. into thymic nude mice. Recombinant BMP-2 also enhanced angiogenesis in Matrigel plugs containing A549 cells in nude mice. The BMP-2 antagonist noggin abrogated BMP-2-induced angiogenic response. Furthermore, antisense transfection of BMP-2 cDNA resulted in a decrease in blood vessel formation in the Matrigel assays. BMP-2 induced tube formation in both human aortic endothelial cells (HAEC) and umbilical vein endothelial cells. BMP-2 also stimulated proliferation of HAEC. The ability of BMP-2 to activate endothelial cells was further demonstrated by its ability to phosphorylate Smad 1/5/8 and ERK-1/2 and to increase expression of Id1. This study reveals that BMP-2 enhanced the angiogenic response in developing tumors. Furthermore, these data suggest that BMP-2 stimulation of angiogenesis may involve the activation of endothelial cells.     

Aluminum impairs gap junctional intercellular communication between astroglial cells in vitro

The purpose of the present study was to investigate the effect of aluminum on gap junctional intercellular communication (GJIC) in cultured astrocytes. In the CNS the extracellular environment and metabolic status of neurons is dependent upon astrocytes, which are known to exhibit GJIC. This cell-to-cell communication provides a cytoplasmic continuity between adjacent cells, allowing exchange of diverse ions, second messengers, and metabolites. To study the effects of aluminum intoxication on GJIC in cultured glial cells, astroglial cell cultures obtained from fetal rat brains were exposed to aluminum lactate for 2-6 weeks. To demonstrate the metabolic coupling of neighboring cells, the technique of microinjection of the gap junction permeable substance neurobiotin was performed. Whereas in controls intensive GJIC was observed by dye transfer of neurobiotin from the microinjected cell into the adjacent astrocytes, aluminum treatment significantly impaired this cellular communication. As aluminum is known to affect cytoskeletal elements, additional investigations into the organization of intermediate filaments (glial fibrillary acid protein, GFAP) and microfilaments in control astrocytes and subsequent aluminum exposure were performed with the aid of fluorescence microscopy and rapid-freeze, deep-etch electron microscopy. Aluminum exposure led to an aggregation of GFAP-positive filaments near to the cell nucleus, accompanied by a destruction of the actin cytoskeleton, especially close to the cell membrane. Ultrastructurally these data could be verified as prominent areas without actin filaments contacting the cell membrane detectable in aluminum-treated astrocytes. Immunohistochemical staining of Cx43 revealed an impaired trafficking of this connexin into the cell prolongations following aluminum treatment, although electron-microscopic data revealed that gap junctions between adjacent astrocytes were still present after aluminum incubation for 24 days. In conclusion, in cultured astrocytes the morphological integrity of microfilaments and the intermediate filament network seem to be fundamental for the translocation of connexins from Golgi complex into the cellular prolongation to exhibit proper and extensive cellular communication through gap junctions.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro

The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.