Hyaluronan and CD44: strategic players for cell-matrix interactions during chondrogenesis and matrix assembly

Embryonic induction, soluble and insoluble factors, receptors, and signal transduction are orchestrated for the morphogenesis of the cartilage elements. The interaction of cells with the extracellular matrix (ECM) may lead to altered cellular response to morphogens based on the formation of new adhesive contacts, or the uncoupling of cell-matrix interactions. Hyaluronan's influence on cell behavior, and its intimate association with cells are accomplished by a wide variety of specific binding proteins for hyaluronan. The temporal expression of the hyaluronan receptor CD44 (which is expressed as several alternatively spliced variants) may be strategic to many of these cell-matrix interactions during chondrogenesis. CD44 expression is temporally coincident with the reduction of intercellular spaces at the regions of future cartilage deposition. The spatial organization of CD44 at the cell surface may function to establish or regulate the structure of the pericellular matrix dependent on a hyaluronan scaffold. As the ECM is modified during embryogenesis, the cellular response to inductive signals may be altered. An uncoupling of chondrocyte-hyaluronan interaction leads to chondrocytic chondrolysis. Thus, consideration of cell-matrix interactions during chondrogenesis, in the light of our current understanding of the temporal and spatial expression of signaling morphogens, should become a promising focus of future research endeavors.     

Expression and modulation of CD44 variant isoforms in humans

CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines.     

CD44 isoforms during differentiation and development

During mouse early development cell adhesion molecules are indispensable for the embryo organisation. A family of molecules probably involved in development is the transmembrane glycoprotein CD44 family, which exists in multiple isoforms. These are generated by alternative splicing of the pre-mRNA, resulting in the enlargement of the extracellular part of the molecule. The standard form of CD44 is widely expressed in adult tissues and in embryos from day 9.5 post coitum onwards, while the numerous variant isoforms exhibit highly specialised patterns of expression that are already in the egg cylinder at day 6.5 of development. In lymphohemopoiesis, specific variant isoforms also emerge at decisive differentiation stages. Although specific ligands for the variant region still await isolation, the highly organised expression of CD44 variant isoforms suggests they have a pivotal role in cellular interactions during early development, pattern formation and hemopoiesis.     

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration

CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.     

Analysis of proteins showing differential changes during ATP oscillations in chondrogenesis

Prechondrogenic condensation is a critical step for skeletal pattern formation. Our previous study showed that ATP oscillations play an essential role in prechondrogenic condensation because they induce oscillatory secretion. However, the molecular mechanisms that underlie ATP oscillations remain poorly understood. We examined how differential changes in proteins are implicated in ATP oscillations during chondrogenesis by using liquid chromatography/mass spectrometry. Our analysis showed that a number of proteins involved in ATP synthesis/consumption, catabolic/anabolic processes, actin dynamics, cell migration and adhesion were detected at either the peak or the trough of ATP oscillations, which implies that these proteins have oscillatory expression patterns that are coupled to ATP oscillations. On the basis of the results, we suggest that (1) the oscillatory expression of proteins involved in ATP synthesis/consumption and catabolic/anabolic processes can contribute to the generation or maintenance of ATP oscillations and that (2) the oscillatory expression of proteins involved in actin dynamics, cell migration and adhesion plays key roles in prechondrogenic condensation by inducing collective adhesion and migration in cooperation with ATP oscillations. Copyright © 2014 John Wiley & Sons, Ltd.     

Temporal expression of CD44 during embryonic chick limb development and modulation of its expression with retinoic acid.

Hyaluronan-cell interactions are initiated co-ordinately with mesenchymal condensation during chondrogenic differentiation in the limb bud. Hyaluronan is responsible for the retention and organization of proteoglycan within the cartilage matrix. Hyaluronan-CD44 binding also retains proteoglycan aggregates to the chondrocyte plasma membrane. A sequence for CD44 protein in chick has recently been reported, but never evaluated in chick chondrocytes. Total RNA was isolated from embryonic chick limb buds, stages 18, 19, 24, 25 and 30. Using semi-quantitative RT-PCR, expression of aggrecan, this chick CD44 orthologue and GAPDH mRNA was analyzed. Aggrecan expression was detected at all stages, but was increased at stage 30. CD44 mRNA was detected at extremely low levels at stage 18 to higher levels in the latter stages. Thus, the temporal expression of CD44 mRNA correlated with the onset of pre-cartilage condensation. The full-length chick chondrocyte CD44 cDNA was obtained following RT-PCR using RNA derived from tibial chondrocytes from stage 37 chick embryos. The nucleotide sequence was used to generate an amino acid sequence and analyses revealed homologies of 44.4% with mouse, 47.8% with bovine and 46.3% with human CD44. Tibial chondrocytes were cultured in the presence or absence of retinoic acid for 36 or 72 h. By RT-PCR, expression of aggrecan and the CD44 mRNA by chick chondrocytes was decreased after retinoic acid treatment, while GAPDH expression showed no change. As expected, control chondrocytes exhibited a round morphology while retinoic acid-treated chondrocytes were elongated. The retinoic acid-treated chondrocytes also exhibited reduced hyaluronan binding. This functional assay indicates a role for a CD44 receptor in matrix retention by chick chondrocytes.     

Human CD8+ T cells display a differential ability to undergo cytokine-driven bystander activation

A subset of CD44^hiCD8⁺ T cells in some, but not all mice, can be induced to rapidly secrete IFNγ during infection with Listeria monocytogenes. This response is dependent on the presence of both IL-12 and IL-18 and does not require engagement of the T cell receptor. In this study, we demonstrate that human CD8⁺ T cells also vary widely in their ability to secrete IFNγ within 15 h of either Listeria infection or cytokine stimulation. The magnitude of the rapid IFNγ response correlated more closely with the intrinsic responsiveness of the T cells to cytokine stimulation rather than the amount of IL-12 produced. CD8⁺ T cells from 2 out of 16 blood donors (12.5%) failed to generate a significant IFNγ response. These results demonstrate that bystander activation of CD8⁺ T cells varies among individuals and validate further study of the differential responses observed using BALB/c vs. C57BL/6 mice.     

Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.     

CD44s and CD44v6 expression in head and neck epithelia

Background

CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.

Methods

Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.

Results

In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.

Conclusion

CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.     

CD44与肿瘤转移

透明质酸受体CD44是一类重要的粘附分子,与肿瘤转移密切相关,。早期认为CD44可促进肿瘤细胞的转移,但近来发现CD44与肿瘤志移之间的关系十分复杂,与CD44的分子类型及肿瘤组织类型皆有关。因此,尚需深入了解CD44在肿瘤转移过程中的分子机制,目前的研究发现CD44可能影响了肿瘤细胞的粘附,运动和胞外基质的降解等过程,临床上CD44有可能成为新的诊断指标和治疗靶点。     

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44.     

E-cadherin negatively regulates CD44-hyaluronan interaction and CD44-mediated tumor invasion and branching morphogenesis

CD44 is a principal cell-surface receptor for hyaluronan (HA). Up-regulation of CD44 is often associated with morphogenesis and tumor invasion. On the contrary, reduction of cell-cell adhesion due to down-regulation of E-cadherin is associated with the invasive and metastatic phenotype of carcinomas. In our current study, we investigated the functional relationship between CD44 and E-cadherin. We established an inverse correlation between CD44 and E-cadherin indicating that the cells expressing higher levels of E-cadherin display weaker binding affinity between CD44 and HA. By using TA3 murine mammary carcinoma (TA3) cells, which display CD44-dependent HA binding, branching morphogenesis, and invasion, we demonstrated an inverse functional relationship between CD44 and E-cadherin by transfecting exogenous E-cadherin into the cells. Our results showed that increased expression of E-cadherin in TA3 cells, but not ICAM-1, weakens the binding between CD44 and HA and blocks spreading of the cells on HA substratum and CD44-mediated branching morphogenesis and tumor cell invasion. The results reported here demonstrated for the first time that E-cadherin negatively regulated CD44-HA interaction and CD44 function and suggested that balanced function of CD44 and E-cadherin may be essential for normal epithelial cell functions, and imbalanced up-regulation of CD44 function and/or down-regulation of E-cadherin function likely contributes to tumor progression.     

CD44 in inflammation and metastasis

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered. Abbreviations: aa, amino acid(s); CS, chondroitin sulfate; CSPG, chondroitin sulfate containing proteoglycan; CD44H, ‘hematopoietic’, also called ‘standard’, isoform of CD44 which contains none of the alternatively spliced variant exons; CD44-Rg, CD44 receptor globulin, a secreted chimaeric protein composed of the external domain of the adhesion receptor CD44 and the hinge, CH2 and CH3 regions of human immunoglobulin-G heavy chain; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate; PB, peripheral blood; PBL, peripheral blood lymphocytes This revised version was published online in November 2006 with corrections to the Cover Date.     

BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF-beta 1 and BMP-2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF-beta 1 and BMP-2 on rat periosteum-derived cells during in vitro chondrogenesis in a three-dimensional aggregate culture. RT-PCR analyses for gene expression of cartilage-specific matrix proteins revealed that treatment with BMP-2 alone and combined treatment with TGF-beta 1 and BMP-2 induced time-dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP-2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF-beta 1 and BMP-2 showed no such expression. Treatment with TGF-beta 1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP-2-treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti-type II and type X collagen antibodies. In contrast, at 14 days, TGF-beta 1 + BMP-2-treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti-type X collagen antibody. Expression of BMPR-IA, TGF-beta RI, and TGF-beta RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP-2, TGF-beta 1 and TGF-beta 1 + BMP-2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP-2 induces neochondrogenesis of rat periosteum-derived cells, and that TGF-beta 1 modulates the terminal differentiation in BMP-2 induced chondrogenesis.