Heterotypic Cell-cell Interactions Research Articles

Heterotypic Cell-Cell Interaction on Micropatterned Surfaces

The aim of this paper was to study the influence of chemical and topographical signals on cell behavior and to obtain a heterotypic cell-cell interaction on microstructured domains. The polysaccharide hyaluronic acid (Hyal) was photoimmobilized on glass surfaces in order to obtain a pattern with squares and rectangles of different dimensions and chemistry. The microstructured surfaces were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The behavior of human coronary artery endothelial cells (HCAEC) and human tumoral dermal fibroblasts (C54) was investigated on these micropatterned surfaces by adhesion studies. Moreover heterotypic interaction among C54 and HCAEC adherent on patterned surfaces was evaluated by time-lapse video microscopy Surface analysis revealed the presence of a pattern consisting of alternating glass and Hyal microstructures whose dimensions decreased from the center to the edge of the sample. Neither HCAEC nor C54 adhered to the immobilized Hyal but both adapted their shape to the different sizes of the glass squares and rectangles. The number of adherent cells depended on the dimensions of both the glass domains and the nuclei of the cells. Co-cultured C54 on HCAEC patterned surfaces showed a heterotypic cell-cell interaction in the same chemical and topographic domain. A heterotypic cell-cell interaction occurred in the same chemical and topographic micro-domains but in narrow areas only. Moreover, the number of cells adhering to the glass domains and cell morphology depended on the dimensions of both adhesive areas and cell nuclei.

Heterotypic interaction of fibroblasts and endothelial cells on restricted area

The polysaccharide hyaluronic acid (Hyal) was photoimmobilized on glass surfaces to obtain a pattern with squares and rectangles of different dimensions and chemistry. The microstructured surfaces were characterized by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Attenuated Total Reflection Infrared Spectroscopy (ATR FT-IR), and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Surface analysis revealed the presence of a pattern consisting of alternating glass and Hyal microstructures whose dimensions decreased from the center to the edge of the sample. The behavior of Human Coronary Artery Endothelial Cells (HCAEC) and human tumoral dermal fibroblasts (C54) was studied on these micropatterned surfaces. Neither HCAEC nor C54 adhered to the immobilized Hyal but both adapted their shape to the different sizes of the glass squares and rectangles. The number of adherent HCAEC and C54 depended on the dimensions of both the glass domains and the nuclei of the cells. Co-cultured C54 on HCAEC patterned surfaces showed a heterotypic cell-cell interaction in the same chemical and topographic domain for the first time. In comparison to other techniques for patterning two different cell types, our approach was non cytotoxic and allowed arbitrary geometric patterns to form on different biocompatible substrata.

Ecto-Phosphorylation of CD98 Regulates Cell-Cell Interactions

Ecto-phosphorylation plays an important role in many cellular functions. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated in vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.

A Subset of Host B Lymphocytes Controls Melanoma Metastasis through a Melanoma Cell Adhesion Molecule/MUC18-Dependent Interaction: Evidence from Mice and Humans

Host immunity affects tumor metastasis but the corresponding cellular and molecular mechanisms are not entirely clear. Here, we show that a subset of B lymphocytes (termed B-1 population), but not other lymphocytes, has prometastatic effects on melanoma cells in vivo through a direct heterotypic cell-cell interaction. In the classic B16 mouse melanoma model, one mechanism underlying this phenomenon is a specific up-regulation and subsequent homophilic interaction mediated by the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule). Presence of B-1 lymphocytes in a panel of tumor samples from melanoma patients directly correlates with MUC18 expression in melanoma cells, indicating that the same protein interaction exists in humans. These results suggest a new but as yet unrecognized functional role for host B-1 lymphocytes in tumor metastasis and establish a biochemical basis for such observations. Our findings support the counterintuitive central hypothesis in which a primitive layer of the immune system actually contributes to tumor progression and metastasis in a mouse model and in melanoma patients. Given that monoclonal antibodies against MUC18 are in preclinical development but the reason for their antitumor activity is not well understood, these translational results are relevant in the setting of human melanoma and perhaps of other cancers.

A new technique for primary hepatocyte expansion in vitro

The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.

EphrinA1 Activates a Src/Focal Adhesion Kinase-mediated Motility Response Leading to Rho-dependent Actino/Myosin Contractility

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell repulsive motility through heterotypic cell-cell interactions. Several Ephs, including EphA2, are greatly overexpressed in certain tumors, in correlation with poor prognosis and high vascularity in cancer tissues. The ability of several Eph receptors to regulate cell migration and invasion likely contribute to tumor progression and metastasis. We report here that in prostatic carcinoma cells ephrinA1 elicits a repulsive response that is executed through a Rho-dependent actino/myosin contractility activation, ultimately leading to retraction of the cell body. This appears to occur through assembly of an EphA2-associated complex involving the two kinases Src and focal adhesion kinase (FAK). EphrinA1-mediated repulsion leads to the selective phosphorylation of Tyr-576/577 of FAK, enhancing FAK kinase activity. The repulsive response elicited by ephrinA1 in prostatic carcinoma cells is mainly driven by a Rho-mediated phosphorylation of myosin light chain II, in which Src and FAK activation are required steps. Consequently, Src and FAK are upstream regulators of the overall response induced by ephrinA1/EphA2, instructing cells to retract the cell body and to move away, probably facilitating dissemination and tissue invasion of ephrin-sensitive carcinomas.

Micropatterning of living cells by laser-guided direct writing: application to fabrication of hepatic–endothelial sinusoid-like structures

Here, we describe a simple protocol for the design and construction of a laser-guided direct writing (LGDW) system able to micropattern the self-assembly of liver sinusoid-like structures with micrometer resolution in vitro. To the best of our knowledge, LGDW is the only technique able to pattern cells "on the fly" with micrometer precision on arbitrary matrices, including soft gels such as Matrigel. By micropatterning endothelial cells on Matrigel, one can control the self-assembly of vascular structures and associated liver tissue. LGDW is therefore uniquely suited for studying the role of tissue architecture and mechanical properties at the single-cell resolution, and for studying the effects of heterotypic cell-cell interactions underlying processes such as liver morphogenesis, differentiation and angiogenesis. The total time required to carry out this protocol is typically 7 h.

Vascular Endothelial Growth Factor Localization in the Adult

Although vascular endothelial growth factor (VEGF) has been well studied in both developmental and pathological angiogenesis, its role in mature blood vessels is poorly understood. A growing body of observations, including the side effects of anti-VEGF therapies as well as the role of soluble VEGFR1 in preeclampsia, points to an important role for VEGF in maintenance of stable blood vessels. To better understand the potential function of VEGF in mature vessels, a survey of VEGF localization in adult mice was conducted. In adult VEGF-lacZ mice, VEGF was expressed in a cell-specific manner by cells overlying fenestrated and sinusoidal blood vessels, including podocytes, choroid plexus epithelium, and hepatocytes, as well as in tissues with high metabolic demands or with secretory functions, such as cardiac and skeletal myocytes, Leydig cells, prostatic epithelium, and salivary serous epithelium. VEGF was not detected in most endothelium but was specifically expressed by aortic endothelial cells where VEGFR2 was found to be phosphorylated, indicating an autocrine loop. Additionally, VEGFR2 was constitutively phosphorylated in the liver, lung, adipose, and kidney in vivo, providing evidence consistent with a role for VEGF in adult tissues. These observations support the concept that VEGF acts in the adult to stabilize mature vessels.

Influence of Umbilical Cord Blood-Derived Endothelial Precursor Cells on a Human Umbilical Vein Endothelial Cell Co-Culture In Vitro Angiogenesis Assay.

To assess in vitro angiogenesis, cellular co-culture assays have been utilized to study adherence, spreading, differentiation and proliferation, and migration of endothelial cells. Formation of tubule or capillary-like networks is influenced by the presence of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) but other factors provided by cell sources and/or direct contact with multiple cell types may facilitate this formation. The hypothesis of this study is that umbilical cord blood (UCB)-derived endothelial precursor cells (EPCs) may influence the formation of human umbilical vein endothelial cell (HUVEC) tubule structures during angiogenesis.Methods: UCB-derived EPCs were isolated from CD133negative cells after a 7-day culture on human fibronectin in EGM-2 media. Tubule formation was evaluated (passage 1–2, 20 x 103 or 2 x 103 cells) by adding HUVECs without or with EPCs to cultures of human bone marrow-derived mesenchymal stromal cells (MSCs) under normoxic (20%) conditions (37°C, 5% CO2, containing VEGF, epidermal growth factor, FGF, insulin-like growth factor, heparin, hydrocortisone, and ascorbic acid in EGM-2 medium) for a 2-week period. HUVECs were added to cultures without or with labeling with Vybrant® CM-DiI which allows the temporal observation of tubule formation progress and cellular incorporation. Final tubule formation was confirmed using a primary anti-CD31 (PECAM) antibody followed by a FITC-conjugated secondary antibody for signal amplification.Results: After 2–4 days, linear aggregates of labeled HUVECs (2-D arrangement) were observed. After 14 days, there was remodeling of HUVECs into the development of a 3D network of linear and branched tubule structures. EPCs facilitated the formation of tubules affecting both the extent of tubule formation and also enhanced proliferation of HUVEC cells. A minority (< 5%) of EPCs were incorporated into developing tubules (estimated using CM-Dil-labeled EPCs). To quantify tubule formation, digital pictures of representative areas of culture wells (2–4/well) were acquired. Using Image Pro Plus software, tubules were quantified using multi-parameter analysis with respect to length, area, and perimeter. The presence of EPCs (equal to the number of added HUVECs) significantly enhanced all parameters. In comparison to control samples, the presence of EPCs increased the area, perimeter and size by 15.2-fold, 3.4-fold, and 3.2-fold, respectively. Confocal microscopy revealed that the co-cultures formed anatamoses, indicating the formation of a connected network.Conclusions: Taken together, these results suggest that the presence of cord blood-derived EPCs facilitate tubule formation and development via a heterotypic cell-cell interaction without integrating into the angiogenic structures. Further studies will evaluate the secretion of cytokines and growth factors.

Modulation ofIn VitroAngiogenesis in a Three-Dimensional Spheroidal Coculture Model for Bone Tissue Engineering

One of the major challenges in tissue engineering of bone substitutes remains vascularization of the transplant. We have developed a three-dimensional collagen-based coculture system to assess interactions between human endothelial cells (hECs) and human osteoblasts (hOBs) in vitro. Human umbilical vein endothelial cells (HUVECs) were grown as three-dimensional multicellular spheroids and seeded in a collagen matrix to assess sprouting of the spheroids, that is, formation of tubelike structures resembling early capillaries. Direct cell contact between hOBs and HUVECs was established by incorporating hOBs into the EC spheroids, thus forming heterogeneous cospheroids. Spatial organization of cospheroids and sprout configuration were assessed by immunohistochemical wholemount staining techniques and confocal laser microscopy. Cumulative sprout length of spheroids was quantitatively analyzed by digital imaging planimetry. In this model HUVECs and hOBs formed heterogeneous cospheroids with distinct spatial organization. The ability of HUVEC spheroids to form tubelike structures on angiogenic stimulation with vascular endothelial growth factor and basic fibroblast growth factor was suppressed in heterogeneous HUVEC/hOB cospheroids. The model system introduced in this study may be useful to assess the mechanisms involved in regulating angiogenesis during bone formation and to further investigate the mechanisms by which heterotypic cell-cell interactions inhibit endothelial tube formation for applications in bone tissue engineering.

Microelectrochemical Approach to Induce Local Cell Adhesion and Growth on Substrates

The nature of an albumin-coated substrate that blocks protein adsorption and cell adhesion was rapidly switched to cell-adhesive by exposure to an oxidizing agent such as HBrO. This finding has enabled cellular pattern drawing even on a single-cell level by closely scanning a microelectrode above the substrate and electrochemically producing the agent at the tip of the electrode. The present microelectrochemical cell patterning is applicable even for a previously cell-patterned substrate and for a grooved substrate. These unique technical features will have impacts on a variety of cell-based studies that require the analysis of heterotypic cell-cell interactions and cellular arrangement on an uneven surface such as semiconductor devices.

The GPIIb/IIIa antagonist eptifibatide markedly potentiates platelet-leukocyte interaction and tissue factor expression following platelet activation in whole blood in vitro

Tissue factor (TF) is the most important initiator of intravascular coagulation. Activated platelets are able to adhere to leukocytes and this heterotypic cell-cell interaction results in a CD62P-dependent TF expression on monocytes. GPIIb/IIIa antagonists are inhibitors of the common pathway of platelet aggregation and they are widely used in patients with acute coronary syndromes undergoing coronary interventions. As GPIIb/IIIa antagonists do not prevent platelet activation we investigated the effect a GPIIb/IIIa antagonist, eptifibatide, on the formation of platelet-leukocyte conjugates and leukocyte TF expression. Flow cytometry was used to detect conjugates and TF. When platelets in citrated human blood were stimulated for 30 min with collagen there was a increase in the number of both neutrophils and monocytes with the platelet-specific antigen CD42a, indicating the formation of platelet-neutrophil (P/N) and platelet-monocyte (P/M) conjugates. P/M formation was associated with about a 2.5-fold increase in TF expression on monocytes, whereas P/N formation changed TF expression neutrophils only by about 10%. Eptifibatide enhanced dose-dependently (0.0625-1.5 w g/ml) both collagen-induced P/M formation and monocyte TF expression. Maximum enhancement by about 60 and 120%, respectively, was observed at 0.5 w g/ml eptifibatide. In contrast, eptifibatide had only a minor effect on P/N formation and no effect on neutrophil TF expression. The augmented P/M formation and monocyte TF expression in the presence of a GPIIb/IIIa antagonist may be relevant to the poor antithrombotic efficiency of oral GPIIb/IIIa antagonists as shown in recent large clinical trials.

Heterotypic Smooth Muscle Cell/Endothelial Cell Interactions Differ between Species

Background. In vitro coculture models have been used to study heterotypic cell-cell interactions. This study was performed to determine if species of cell origin affects heterotypic smooth muscle cell (SMC) endothelial cell (EC) interactions in coculture.Methods. To study the effect of ECs on SMC proliferation, ECs were cultured on porous Dacron membranes. SMCs were added opposite the ECs or on bare membranes on Day 3, and after 4 days, cells were harvested for cell counts. To study the effect of SMCs on EC proliferation, ECs at a density of 5 × 105 cells/membrane were added to bare membranes or on membranes opposite SMCs plated 2 days earlier. After 48 h, cells were harvested for cell counts. (N = 3/condition, experiments repeated ×2.) Cells of human and bovine aortic origin were used.Results. The effect of coculture on cell growth differed between species. The effect of heterotypic interactions between human cocultured cells was coinhibitory on the rate of growth as compared to the growth of cells cultured alone. Growth of cocultured ECs was 55.2 ± 8.7% less than that of ECs cultured alone while growth of cocultured SMCs was 27.2 ± 6.0% less than growth of SMCs cultured alone. This contrasted with the bovine EC stimulation of SMC proliferation, with 66.8 ± 5.0% greater growth of cocultured SMCs compared to SMCs cultured alone, and failure of bovine SMCs to decrease EC proliferation.Conclusions. Since significant differences in cell-material interactions occur in vivo between species, the finding that in vitro heterotypic cell-cell interactions are species dependent is not surprising. This fundamental difference in cell behavior stresses the potential importance of using human cells in studies evaluating cell-cell and cell-material interactions in vitro.

Using electroactive substrates to pattern the attachment of two different cell populations.

This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.

Models for the study of uterine receptivity for blastocyst implantation.

A variety of models have been developed to study endometrial receptivity which involves normal, appropriately timed endometrial development and remodeling for blastocyst attachment and trophoblast invasion during the luteal phase of the menstrual cycle. Due to species differences, the human is by far the best model per se by which to study human endometrial receptivity. Techniques have evolved to obtain in vivo data on endometrial receptivity using hysteroscopy, ultrasonography or magnetic resonance imaging. Despite species differences, comparative studies of mammalian models and tissue- and cell culture models using endometrial tissue or cells harvested at particular phases of the reproductive cycle, or following experimental manipulation, have been used productively to study endometrial function. Differences as well as similarities have proven to be instructive. Such models have been used to study a variety of entities, such as homotypic and heterotypic cell-cell interaction, the role of steroids, cytokines, growth factors, immunomodulatory agents and pharmacological substances. These models have also been used to study cellular, biochemical and molecular mechanisms involved with uterine receptivity. This chapter was designed to provide a critical review of contemporary literature relating to in vivo models and laboratory strategies and paradigms for the study of uterine receptivity for blastocyst implantation.

Osteoprogenitor cell frequency in rat bone marrow stromal populations: Role for heterotypic cell-cell interactions in osteoblast differentiation

Glucocorticoids, notably dexamethasone (Dex), have been reported to be a requirement for osteoprogenitor cell differentiation in young adult rat bone marrow stromal cell populations. We have reinvestigated the requirement for Dex and analyzed the frequency of osteoprogenitor cells present. Stromal cells were grown as primary or first subcultures in the presence or absence of Dex and their expression of osteogenic markers (alkaline phosphatase activity, hormone responsiveness, and matrix molecules, including type I collagen, osteopontin, bone sialoprotein, and osteocalcin), as well as their functional capacity to differentiate to form a mineralized bone nodule, were assessed. Dex increased, but was not an absolute requirement for, the expression of osteogenic markers. Bone nodule formation was plating cell density dependent and occurred under all combinations of treatment with or without Dex but was maximal when Dex was present in both the primary and secondary cultures. Dex increased CFU-F by approximately 2-fold, but increased CFU-O (osteoprogenitor cells; bone nodule forming cells) by 5- to 50-fold depending on the cell density and duration of treatment. Neither CFU-F nor CFU-O expression followed a linear relationship in limiting dilution analysis until very high cell densities were reached, suggesting cooperativity of cell types within the population and a multitarget phenomenon leading to osteoprogenitor differentiation. When a large number of nonadherent bone marrow cells or their conditioned medium was added to the stromal cells, osteoprogenitors comprised approximately 1/100 of plated adherent cells and their expression followed a linear, single-hit relationship. By contrast, rat skin fibroblasts or their conditioned medium totally inhibited bone nodule formation. These data support the hypothesis that in marrow stroma, as in other bone cell populations such as those from calvaria, there are at least two classes of osteoprogenitor cells: those differentiating in the absence of added glucocorticoid and those requiring glucocorticoid to differentiate, that more than one cell type is limiting for stromal osteoprogenitor differentiation suggesting a role for heterotypic cell-cell interactions in osteogenesis in this tissue, and that Dex may be acting directly and/or indirectly through accessory cells in the bone marrow to alter osteoprogenitor cell expression.

Direct Interaction between Gingival Fibroblasts and Lymphoid Cells Induces Inflammatory Cytokine mRNA Expression in Gingival Fibroblasts

In inflamed periodontal lesions, dense infiltration of lymphocytes is usually observed in the extravascular periodontal connective tissue, adjacent to gingival fibroblasts. Our previous study revealed that activated lymphocytes can adhesively interact with gingival fibroblasts in vitro. In the present study, we investigated whether gingival fibroblasts are activated through direct interaction with lymphoid cells by monitoring the expression of inflammatory cytokine mRNA in human gingival fibroblasts (HGF). Co-culture with various human lymphoid cells in vitro resulted in a marked increase in the expression of IL-1alpha, IL-1beta, and IL-6 mRNA by the HGF. In addition, expression of the mRNA of the IL-1beta-converting enzyme (ICE), which is essential to produce the mature form of IL-1beta, was constitutively observed in the HGF, suggesting that mature IL-1beta is produced by these cells. When HGF were cultured with the culture supernatant of the lymphoid cells, the increase in the inflammatory cytokine mRNA expression was not observed. Similarly, when HGF and lymphoid cells were cultured in the same well but separated by a membrane which prevented direct contact between the cells, no increase in inflammatory cytokine mRNA expression was observed. These results strongly indicate that direct interaction between these heterotypic cell types transduces activation signals into HGF that induce an increase in inflammatory cytokine mRNA expression. Furthermore, IL-1beta mRNA expression in the HGF was synergistically increased when HGF directly interacted with lymphoid cells in the presence of exogeneous IL-1beta. The present study demonstrates that direct interaction between HGF and lymphoid cells stimulates HGF to increase inflammatory cytokine mRNA expression, and raises the possibility that heterotypic cell-cell interaction may facilitate local inflammatory reactions.

Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation.

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell-cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell-cell contact caused evident morphological changes accompanied with tight cell-cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow-derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.

CD44 isoform expression in periodontal tissues: cell-type specific regulation of alternative splicing.

CD44 functions as a receptor for various extracellular matrices and plays crucial roles in homotypic and heterotypic cell-cell interactions. Recently, the molecular structure of CD44 has been extensively analyzed and multiple isoforms produced by alternative splicing of messenger RNA have been identified. In this study, we examined the expression of CD44 isoforms on different cell types isolated from periodontal tissue. In order to examine tissue differences in CD44 isoform expression, we established in vitro cell culture of human gingival fibroblasts (HGF), human periodontal ligament cells (HPDL) and human gingival epithelial cells (HGEC). These cells all expressed CD44 protein and messenger RNA. However, immunoprecipitation and Northern blot analysis revealed that HGEC expressed larger CD44 isoforms than HGF and HPDL. Reverse transcription-polymerase chain reaction with primers flanking the insertion site of alternatively spliced exons was used to study details of the heterogeneity. All cells examined expressed a major band in the absence of alternatively spliced exons and additional larger bands. In particular, HGEC contained more abundant high molecular mass species. In vitro stimulation by IL-1 beta, TNF alpha or phorbol 12-myristate 13-acetate induced an increase in total CD44 messenger RNA in HGF but not change in overall patterns of CD44 isoform expression. However, the isoform expression of HGEC was sensitive to cell density. The amount of larger isoform was decreased by culturing cells beyond confluence. These findings suggest that CD44 isoform expression is cell type-specifically regulated in periodontium and altered according to growth phase of HGEC.

N3 10:45 Thymic Heterotypic cell-cell interactions in gene-targeted mutant mice

N3 10:45 Thymic Heterotypic cell-cell interactions in gene-targeted mutant mice