The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10)

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.     

Biological activities of laminin

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.     

Deposition of Basement Membrane Proteins in Attachment and Neurite Formation of Cultured Murine C-1300 Neuroblastoma Cells

The deposition of the basement membrane glycoproteins, laminin, fibronectin, and type IV procollagen was studied by indirect immunofluorescence microscopy during the attachment and differentiation of murine C-1300 neuroblastoma cells. A typical cytoplasmic perinuclear staining for the basement membrane antigens was seen both in undifferentiated and differentiated cells. Freshly seeded suspended cells lacked surface fluorescence but in two hours after plating, distinct punctate laminin deposits became discernible on the ventral surface of the cells. Notably, in sparsely seeded undifferentiated cultures, the cell-associated extracellular laminin deposits could only be detected under the primary attaching cells, whereas daughter cells in clonal cell colonies lacked such fluorescence. In cultures induced to neurite formation with dibutyryl cyclic AMP, laminin deposition was also detected in association with the growing cytoplasmic extensions. No distinct differences were found between the secreted proteins of cultures of differentiated and nondifferentiated neuroblastoma cells, but the patterns of fucosylation of high-molecular weight proteins in the two cultures were markedly different. We conclude that cultured neuroblastoma cells both synthesize, secrete and deposit laminin. The distribution of laminin during neuroblastoma cell attachment and neurite extension suggests that this glycoprotein may be involved in cell–to–substratum interactions in C-1300 cell cultures.     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

Automated technique to evaluate thymocyte adhesion to thymic epithelial cells

Thymocyte adhesion to thymic epithelial cells is a relevant issue during intrathymic T-cell differentiation, and directly intervenes in the generation and expansion of the T-cell repertoire. In view of these data, it was apparent the usefulness of an automated strategy to evaluate the degree of thymic epithelial cell-thymocyte adhesion. This prompted us to develop an ELISA procedure (using an anti-Thy1 reagent) to determine the degree of thymocyte adhesion onto cultured thymic epithelial cells. The procedure described herein is simple, non-radioactive and reproducible. Additionally, it can potentially be applied to quantitate the degree of thymocyte adhesion to any cellular or non-cellular substrate (for example, extracellular matrix). Moreover, it detected fluctuations of thymocyte adhesion secondary to glucocorticoid treatment of epithelial cells. Thus, it can be regarded as a further tool to analyze intrathymic interactions.     

A Lymphocyte Serine Protease Granzyme A Causes Detachment of a Small-Intestinal Epithelial Cell Line (IEC-6)

Granzyme A (GzmA) is a serine protease (trypsin-like specificity) produced in cytotoxic lymphocytes. This enzyme is believed to enter virus-infected cells and growing tumors and induce apoptosis, but the roles of GzmA expressed in lymphocytes scattered through the epithelial layer of the normal small intestine are unknown. In the present study, recombinant rat GzmA (rGzmA) was found to cause morphological changes and detachment of a non-transformed rat small-intestinal epithelial cell line IEC-6, although the rGzmA-treated cells detached as aggregates with no changes characteristic of apoptosis. rGzmA-induced deformation and detachment occurred in IEC-6 cells plated with collagen type IV and fibronectin, but not in those plated with laminin. These findings suggest that GzmA in the normal small intestine participates in the reduction of adhesion between epithelial cells and basement membranes, through its ability to cleave extracellular matrix components.     

SPARC/Osteonectin Functions to Maintain Homeostasis of the Collagenous Extracellular Matrix in the Periodontal Ligament

Expression of secreted protein acidic and rich in cysteine (SPARC)/osteonectin, a collagen-binding matricellular protein, is frequently associated with tissues with high rates of collagen turnover, such as bone. In the oral cavity, expression of SPARC/osteonectin has been localized to the periodontal ligament (PDL), a collagen-rich tissue with high rates of collagen turnover. The PDL is critical for tooth position within the alveolar bone and for absorbing forces generated by chewing. To characterize the function of SPARC/osteonectin in PDL, SPARC/osteonectin expression in murine PDL was evaluated by immunochemistry at 1, 4, 6, and >18 months. Highest levels of SPARC/osteonectin were detected at 1 and >18 months, with decreased levels associated with adult (4–6 months) PDL. To determine whether the absence of SPARC/osteonectin expression influenced cellular and fibrillar collagen content in PDL, PDL of SPARC-null mice was evaluated using histological stains and compared with that of wild-type (WT). Our results demonstrated decreased numbers of nuclei in PDL of SPARC-null mice at 1 month. In addition, decreased collagen volume fractions were found at 1 and >18 months and decreases in thick collagen fiber volume fraction were detected at 4, 6, and >18 months in SPARC-null PDL. The greatest differences in cell number and in collagen content between SPARC-null and WT PDL coincided with ages at which levels of SPARC/osteonectin expression were highest in WT PDL, at 1 and >18 months. These results support the hypothesis that SPARC/osteonectin is critical in the control of tissue collagen content and indicate that SPARC/osteonectin is necessary for PDL homeostasis. (J Histochem Cytochem 58:871–879, 2010)     

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9′10), (ii) an Arg to Ala synergy site mutation (FnIII9^R→^A10), (iii) a two-Gly (FnIII9^2G10) insertion, and (iv) a four-Gly (FNIII9^4G10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9′10 and FnIII9^2G10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9′10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9′10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

回转对离体大鼠成骨细胞中骨粘连蛋白及骨桥素mRNA的影响

为研究模拟失重对成骨细胞细胞外基质mRNA的影响,实验采用离体人鼠成骨细胞水平轴回转模拟失重效应,用RT-PCR技术分别检测成骨细胞中骨桥素(osteopontin,OPN)及骨粘连蛋白(osteonectin,ON)mRNA的水平,并观察细胞培养液中碱性磷酸酶(alkaline phosphatase,ALP)的活性和骨钙素(osteocalcim, BGP)含量的变化。结果观察到,分别回转24、48、72h后,OPN、ON的mRNA含量及细胞培养液中BGP含量均显著下降,细胞培养液中ALP活性也呈下降趋势。上述结果表明,模拟失重后成骨细胞OPN及ON的表达下调,进而使BGP及ALP的分泌量减少,从而导致骨钙化能力降低,提示模拟失重导致的细胞外基质蛋白基因表达下降可能是模拟火重引起骨丢失的原因之一。     

Human SY5Y Neuroblastoma Cell Interactions with Laminin Isoforms: Neurite Outgrowth on Laminin-5 Is Mediated by Integrin α3β1

Laminin (Ln) isoforms may play important roles in neuronal development, particularly axon guidance, but neural receptors mediating interactions with Ln are not entirely understood. In this paper, we have compared the adhesive and process outgrowth activities of a human neuroblastoma cell line SY5Y on various laminin isoforms. Cell adhesion and process outgrowth were examined on murine Ln-1 (Englebreth-Holm-Swarm sarcoma laminin), human placental Ln-1 (human Ln-1[p]), human Ln-2 (merosin), human Ln-5 (kalinin/epiligrin/nicein), and human foreskin keratinocyte extracellular matrix extract (human HFK-ECM). Ln-5 was shown to evoke process outgrowth in amounts comparable to other Ln isoforms. Antibody perturbation experiments showed that adhesion and process outgrowth on murine Ln-1 was primarily mediated by the integrin α1β1, whereas adhesion and outgrowth on human Ln-5 and human HFK-ECM were mediated by α3β1. Adhesion to human Ln-1(p) and Ln-2 was not blocked by addition of anti-α1 or anti-α3 antibodies alone, but adhesion was partially perturbed when these antibodies were added in combination. Process outgrowth on human Ln-1(p) was blocked when either anti-α3 or anti-β1 antibodies were added, indicating that α3β1 is the primary integrin heterodimer responsible for process extension on this substrate. These results demonstrate that Ln-5 and other Ln isoforms support comparable levels of adhesion and process outgrowth, but different integrin heterodimers, alone and in combination, are used by SY5Y cells to mediate responses.     

Rat Sertoli cell aromatase cytochrome P450: Regulation by cell culture conditions and relationship to the state of cell differentiation

Summary Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions. No differences were found in P450arom enzymatic activity measured by the³H₂O release method in microsomes prepared from Sertoli cells cultured under the various conditions. Similarly, no differences were observed in the amount of protein detected by immunoblot analysis of Sertoli cell extracts using an antiserum raised against the human placental enzyme. Recombination experiments using microsomes from cells cultured on plastic or in the chambers and cytosol from control or FSH-treated cells cultured on plastic also proved inadequate in inducing P450arom activity. These data suggest that: a) P450arom activity could be used as a specific marker for Sertoli cell differentiation, and b) the differentiation process in Sertoli cells is associated with specific changes in the microenvironment or the regulation of P450arom, or both, that rendered the enzyme insensitive to FSH or cAMP induction.     

Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth

Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.