Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development

 Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7–9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7–8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events. Accepted: 13 January 1998     

CD44 variant isoforms associate with tetraspanins and EpCAM

The metastasizing subline of the rat pancreatic adenocarcinoma BSp73 expresses a set of membrane molecules, the combination of which has not been detected on non-metastasizing tumor lines. Hence, it became of interest whether these molecules function independently or may associate and exert specialized functions as membrane complexes. Separation of CD44v4-v7 containing membrane complexes in mild detergent revealed an association with the alpha3 integrin, annexin I, EpCAM, and the tetraspanins D6.1A and CD9. EpCAM and the tetraspanins associate selectively with CD44 variant (CD44v), but not with the CD44 standard (CD44s) isoform. The complexes are found in glycolipid-enriched membrane (GEM) microdomains, which are dissolved by stringent detergents, but the complexes are not destroyed by methyl-beta-cyclodextrin (MbetaCD) treatment, which implies that complex formation does not depend on a lipid-rich microenvironment. However, a complex-associated impact on cell-matrix and cell-cell adhesion as well as on resistance towards apoptosis essentially depended on the location in GEMs. Thus, CD44v-specific functions may well be brought about by complex formation of CD44v with EpCAM, the tetraspanins, and the alpha3 integrin. Because CD44v4-v7-EpCAM complex-specific functions strictly depended on the GEM localization, linker or signal-transducing molecules associating with the complex are likely located in GEMs.     

Expression of CD44 isoforms during bleomycin-or radiation-induced pulmonary fibrosis in rats and mini-pigs

The distribution of CD44s and CD44v molecules in normal and injured lung tissue of rats and minipigs was studied by examining the immunohistochemical binding of monoclonal antibodies against CD44 isoforms. We showed that the expression of CD44v and CD44s varies greatly among different pulmonary fibrosis samples and that some tissues express either enhanced expression of CD44s, particularly in the interstitium and on alveolar macrophages, or very low levels of CD44v in the alveolar epithelium. Normal type II pneumocytes expressed the CD44s and CD44v molecules at the basolateral aspect of the cell. Such localisation favours a role for CD44 in epithelial cell-fibroblast interaction during lung development and repair.     

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.     

Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.     

Modulation of neuronal differentiation by CD40 isoforms

Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40^−/− deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40^−/− mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may represent important therapeutic modalities for neurodegenerative and neurodevelopmental disorders, as well as, for enhancement of neurogenesis.     

CD44 clustering is involved in monocyte differentiation

Differentiation of monocytes into macrophages is an import ant process under physiological and pathological conditions, but the underlying mechanism of monocyte differentiation is not completely clear. Some adhesion molecules have been reported to play an important role in cell differentiation. CD44 is an important adhesion molecule that mediates cell cell and cellmatrix interaction, and participates in a wide variety of cellular functions. As CD44 has been reported to show different activated states between monocytes and macrophages, we propose that CD44 may be involved in monocyte differentiation. In this study, we explored the role of CD44 in monocyte differentiation and further studied the mechanisms that were involved in. THP1 cells （human monocyfic leukemia cell line） were induced with phorbol 12myristate 13acetate （PMA） to establish the model of monocyte differentiation in vitro. It was found that CD44 expression and binding capacity to hyaluronic acid were increased significantly, and the distribution of CD44 was con verted into clusters during differentiation. The PMAinduced CD44 clustering and CD44 high expression were suppressed by blocking CD44, which resulted in the inhibition of CD14 expression. PMAinduced phosphorylation of ERK1/2 signal was also suppressed by blocking CD44. Our results suggested that CD44 was involved in monocyte differentiation. The mechanisms of monocyte differentiation following CD44 acti vation may include CD44 high expression and clustering which in turn lead to phosphorylation of ERK1/2.     

Growth and differentiation regulate CD44 expression on human keratinocytes

Summary Several members of the CD44 family of hyaluronan receptors are expressed on keratinocytes. To identify factors that might be important in regulating CD44 expression, we studied CD44 expression on keratinocytes growing in vitro under a variety of conditions and on cells isolated directly from epidermis. Using Western immunoblots and metabolic labeling, we showed that the pattern of CD44 proteins expressed by keratinocytes was strongly influenced by growth and differentiation. Many protein forms of CD44 are expressed on proliferating keratinocytes in preconfluent cultures, whereas only a few forms are expressed on differentiated cells and in confluent cultures. In preconfluent monolayers, at least four splice variants were identified, including epican, CD44H, CD44E, and a 180-kDa variant. In differentiated cells or in confluent cultures, by contrast, only epican and the 180-kDa protein variant were found. Synthesis of all variants is strongly downregulated when keratinocytes become confluent or when they differentiate. Epican is the predominant form of CD44 on keratinocytes under all conditions and is expressed as a heparan, chondroitin, or keratan sulfate proteoglycan. Preconfluent basal keratinocytes, but not confluent or differentiated keratinocytes, also express chondroitin sulfate proteoglycan forms of CD44E and of the 180-kDa core protein. The modal size of the epican expressed on differentiated keratinocytes is smaller than the size of the epican expressed on basal keratinocytes. Thus, cell confluence and differentiation regulate several aspects of CD44 expression on keratinocytes, suggesting nuances in function for the different protein forms.     

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.     

Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44

Correlations have been noted between the expression of certain alternatively spliced CD44 isoforms and the metastatic propensity of various histologically distinct tumor cell types. The precise mechanism by which particular CD44 isoforms contribute to the metastatic process is, however, unclear. In the present study we demonstrate that CD44R2, a CD44 isoform highly expressed on activated and transformed hemopoietic cells, can recognize and bind a common determinant present on CD44H and CD44R1. Importantly, CD44H lacked this activity. Pretreatment of TIL1 cells expressing CD44H or CD44R1 with chondroitinase ABC inhibited adhesion to CD44R2, suggesting that the unique inserted region present within the CD44R2 molecule, encoded by exon v10, mediates cell adhesion by potentiating the recognition of chondroitin sulfate moieties presented in association with other CD44 molecules. These data help explain the differential involvement of v10-containing CD44 isoforms in tumor metastasis.     

CD44 variant,but not standard CD44 isoforms,mediate disassembly of endothelial VE-cadherin junction on metastatic melanoma cells

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8-v10 induced junction disassembly and vascular endothelial (VE)-cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)-mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8-v10 promoted endothelial junction disruption. Overexpression of CD44v8-v10 but not of standard CD44 (CD44s) promoted gap formation in the non-metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE-cadherin mutant Y658E supported transmigration of CD44-silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.     

Mevinolin enhances osteogenic genes (ALP,type I collagen and osteocalcin), CD44, CD47 and CD51 expression during osteogenic differentiation

AimsIn this study, we evaluated the effect of mevinolin on the expressions of osteogenic genes and surface molecules expression during osteogenesis.Main methodsD1 cells were cultured in osteogenic differentiation medium (ODM) for 6 days, treated with mevinolin for 2 days, and then subjected to alizarin red S staining, MTT assays, alkaline phosphatase (ALP) activity determinations, energy dispersive X-ray spectrophotometry (EDX), real-time PCR, Western blot, fluorescence microscopy and FACS analysis.Key findingsMevinolin is commonly prescribed and widely used to lower cholesterol levels, and offers an important, effective approach to the treatment of hypercholesterolemia and arteriosclerosis. However, the direct effect of mevinolin on osteogenesis in vitro has not been clarified. ODM has been previously shown to increase the osteoblast differentiation of D1 cells. In the present study, we investigated the expressions of osteogenic genes and surface molecules during osteoblast differentiation induced by mevinolin. We found that the induction of ALP, type I collagen, osteocalcin, CD44, CD47 and CD51 by mevinolin is responsible for the osteoblastic differentiation of D1 cells.SignificanceOur data show that mevinolin enhances the expressions of proteins and surface molecules related to osteogenesis.     

Hyaluronidases and CD44 undergo differential modulation during chondrogenesis

Hyaluronan, a high-molecular-weight glycosaminoglycan of cartilage, is deposited directly into the extracellular space by hyaluronan synthases, while hyaluronan catabolism is mediated by the hyaluronidases. An in vitro cell culture system has been established in which human dermal fibroblasts are induced to undergo chondrogenesis. Here, we describe the differential modulation of the hyaluronidases and the up-regulation of the hyaluronan receptor, CD44, during such chondrogenesis. Dermal fibroblasts, plated in micromass cultures in the presence of lactic acid and staurosporine for 24 h, were then placed in serum-free, chemically defined medium. At 3 days, RNA was extracted and RT-PCR performed using primers for the hyaluronidase genes. Marked increase in HYAL1 expression was observed, with only moderate increases occurring in HYAL2 and HYAL3. No expression of HYAL4 and PH-20, the sperm-associated hyaluronidase, was detected. RNA levels correlated well with changes in hyaluronidase enzyme activity. Finally, greater expression and staining for the hyaluronan receptor, CD44s, the standard form, were detected. Differential expression of the somatic hyaluronidases and CD44-mediated hyaluronan turnover play a critical role in cartilage development from mesenchymal precursors.     

Regulation of CDPK isoforms during tuber development

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.     

A novel antiapoptotic mechanism based on interference of Fas signaling by CD44 variant isoforms

There is growing evidence that one of the central common characteristics of tumor and inflammatory cells is their resistance to programmed cell death. This feature results in the accumulation of harmful cells, which are mostly refractory to Fas (FAS, APO-1)-mediated apoptosis. A molecule found on these cells is the transmembrane receptor CD44 with its variant isoforms (CD44v). The establishment of transfectants expressing different CD44v isoforms allowed us to demonstrate that the CD44v6 and CD44v9 isoforms exhibit an antiapoptotic effect and can block Fas-mediated apoptosis. Moreover, we observed that CD44v6 and CD44v9 colocalize and interact with Fas. Importantly, an anti-CD44v6 antibody can abolish the antiapoptotic effect of CD44v6. These results are the first to show that CD44v isoforms interfere with Fas signaling. Our findings improve the understanding of the pathogenesis of cancer and autoimmunity and open new strategies to treat such disorders.     

CD44 variant isoforms are preferentially expressed in basal epithelia of non-malignant human fetal and adult tissues

CD44 is a transmembrane glycoprotein, which can exist in a multitude of isoforms due to alternative splicing of the pre-mRNA. We have generated monoclonal antibodies to several of these variant regions, which are encoded by 10 additional exons in the extracellular part of the molecule. CD44 variant isoforms have been reported to be involved in the malignant progression of rat and human tumours. The precise localization of CD44 variant isoforms in normal developmental and morphogenetic processes is essential for diagnostic studies of human tumorigenesis. Therefore, we have analysed a large number of different human tissues by immunohistochemistry for the expression of CD44 isoforms containing either exons 4v, 6v or 9v. Expression of exon 9v-isoforms was detected in almost all epithelia analysed, with a few exceptions. Exon 6v isoforms are expressed only in squamous and glandular epithelia, e.g. skin epidermis, sweat and sebaceous glands, oesophagus, ducts of the mammary gland, salivary and prostate glands. Detection of exon 4v-encoded isoforms was restricted to the epidermis and the oesophagus. Similar tissue distributions of CD44 variant isoforms were observed in 10-week-old fetal tissues. Since one of the ligands of CD44 is hyaluronic acid (HA), we also analysed the tissue distribution of HA synthetase. HA synthetase was detected in all tissues analysed, showing good correlation with the expression of the standard form of CD44, CD44s.