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PubMed1997

Upregulated renal tubular CD44, hyaluronan, and osteopontin in kdkd mice with interstitial nephritis.

BACKGROUND: The hyaluronan (HA) receptor CD44 is upregulated on parenchymal cells in various inflammatory lesions and could play a role in immune injury. The purpose of the present study was to examine CD44 and its ligands HA and osteopontin (Opn) in a murine model of tubulointerstitial nephritis (TIN). METHODS: The expression of CD44 was investigated by immunofluorescence staining and RNA analysis in kidneys of kdkd mice with autoimmune TIN. The CD44 expression was then correlated with the location of its ligands HA and Opn. RESULTS: CD44 is expressed de novo by tubular epithelial cells (TEC) in areas of tubular injury in kdkd kidneys, but not in normal control kidneys. CD44 positive lymphocytes and macrophages also infiltrate the kidney to kdkd mice. RT-PCR and Southern blot analysis demonstrate that transcripts encoding standard and variant forms of CD44 are increased in kdkd mice with TIN. In parallel the CD44 ligand HA also accumulates in kdkd kidneys in the interstitial space, particularly in cortical areas of tubular injury. Furthermore, the expression of the chemotactic protein Open is enhanced in kdkd kidney, predominantly in areas of tubular injury. Opn mRNA expression also increases markedly in kdkd kidneys compared with normal kidneys, and correlates with disease severity. CONCLUSIONS: Prominent CD44 expression by TEC in areas of tubulointerstitial lesions is a characteristic feature of kdkd mice. The de novo appearance of CD44 on injured TEC might allow interaction with the ligands HA and Opn in vivo. Interaction of CD44 with these ligands could participate in the tubulointerstitial inflammatory response in kdkd mice.

Animals↗
PubMed2005

Pre-transplant plasma and cellular levels of CD44 correlate with acute renal allograft rejection.

BACKGROUND: Since CD44 is involved in activation, proliferation, rolling and extravasation of lymphocytes, we hypothesized that it could be involved in the pathophysiology of acute renal allograft rejection. METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) were collected from patients 24 h prior to transplantation and analysed retrospectively. Soluble CD44, interleukin-2 receptor (IL-2R), intracellular adhesion molecule-1 (ICAM-1) and C-reactive protein (CRP) in plasma were determined by enzyme-linked immunosorbent assay (ELISA). Cellular CD44 expression on peripheral lymphocytes was determined by flow cytometric analysis. RESULTS: Patients who later developed renal allograft rejection had statistically significantly increased soluble CD44 levels, but not soluble ICAM-1, IL-2R or CRP in plasma prior to transplantation. In addition, cellular CD44 on T-lymphocytes was decreased 24 h prior to transplantation in patients that would reject their allograft, compared with patients without rejection. Additionally, plasma CD44 and cellular CD44 revealed an inversely proportional correlation. Lipopolysaccharide (LPS)-induced immune activation did not influence plasma or cellular CD44 levels in healthy volunteers, suggesting that more specific factors influence the shedding of CD44 on T lymphocytes, leading to increased risk of renal allograft rejection. CONCLUSION: Although the exact mechanism remains to be elucidated and further research is required, soluble CD44 levels and cellular surface CD44 on T lymphocytes prior to transplantation might be useful as predictive markers for the occurrence of acute renal rejection.

Acute Disease↗
PubMed1995

Differential expression of CD44 in malignant cutaneous epithelial neoplasms.

The CD44 antigen (ECMRIII, Hermes antigen) is a highly glycosylated cell-surface polypeptide involved in diverse cellular functions, including cell adhesion and lymphocyte-homing receptor activity. CD44 is also expressed in vivo by several tumors, including astrocytomas, meningiomas, and colonic adenocarcinomas. In addition, it has been shown that expression of CD44 appears to confer metastatic potential to cell lines derived from certain adenocarcinomas. In the skin, CD44 is normally expressed in epidermal keratinocytes and hair follicular, sebaceous, and eccrine epithelial cells. However, there have been few data with regard to the expression in vivo of CD44 in primary cutaneous neoplasms. Furthermore, there have not been studies in vivo of the possible differential expression of CD44 in primary versus metastatic tumors in the skin. We have examined by immunohistochemistry the expression of CD44 in cutaneous invasive and metastatic squamous cell carcinomas, metastatic adenocarcinomas, and basal cell carcinomas. All invasive and metastatic squamous cell carcinomas, as well as metastatic adenocarcinomas, strongly expressed CD44; however, basal cell carcinomas were nonreactive or showed only focal, minimal reactivity. Adjacent normal skin demonstrated CD44 immunoreactivity throughout the epidermis, including the basal layer. In addition, hair follicles and sebaceous and eccrine glands expressed CD44. The results suggest that expression of CD44 in these cutaneous epithelial tumors is not related to malignant transformation, but instead may be related to tumor progression and the ability to metastasize.

Adenocarcinoma↗
PubMedDate not supplied

CD44 expression in neuroblastoma and related tumors.

PURPOSE: A retrospective study was conducted to investigate the relationship between CD44 expression in neuroblastoma and related tumors and other known prognostic indicators. MATERIALS AND METHODS: Immunostaining of CD44 was done on surgical specimens of 55 cases (42 patients) of neuroblastoma (NB) and ganglioneuroblastoma (GNB) and nine cases of ganglioneuroma. The percentage of positive tumor cells was scored semiquantitatively (0-4+) by two observers. CD44 expression was then correlated with survival, age, stage, and N-myc amplification. RESULTS: Fifty-seven percent of the patients with NB or GNB had heterogeneous positive staining (2-4+) on their diagnostic specimens. Twenty-four percent of the patients had no staining for CD44, and 19% had 1+ staining. In the 17 cases with N-myc analysis, an inverse relationship was demonstrated between N-myc and CD44 expression by univariate analysis. Lack of expression of CD44 was highly associated with poor survival (p = 0.0002). When assessing the joint effects of age, stage, and CD44 in multivariate analysis, the effect of CD44 remains significant (p = 0.028) and appears to be independent of age and stage. CONCLUSION: Our data suggest a relationship between CD44 and N-myc amplification. Absence or low expression of CD44 correlates with poor survival and may be a biologic marker of tumor aggressiveness. CD44 appears to be an independent prognostic marker and deserves continued investigation in prospective studies of neuroblastoma.

Adolescent↗
PubMed2005

Substance P induced changes in CD74 and CD44 in the rat bladder.

PURPOSE: Substance P (SP) induces rat bladder inflammation along with release of the proinflammatory cytokine, macrophage migration inhibitory factor (MIF). To describe the mechanism of MIF action we examined changes in the amount of CD74 (membrane receptor for MIF), CD44 and phospho-(p-ERK)1/2 in the bladder. MATERIALS AND METHODS: In anesthetized rats the bladder was isolated by cutting the ureters and urine was replaced by saline as intraluminal fluid (ILF). One hour after subcutaneous SP (40 mug/kg) or saline administration the ILF and bladder were collected. Bladder tissue was analyzed for CD74 and CD44 by immunohistochemistry. Western blot analysis determined the relative amounts of bladder tissue MIF, CD74, CD44 and p-ERK1/2. ILF immunoprecipitation followed by Western blot analysis was performed to identify an association of MIF with CD74 and/or CD44. RESULTS: SP induced significant MIF release from the bladder and increased CD74 and CD44 bladder immunostaining. SP treatment increased the total amount of bladder CD74 protein and mRNA, intracellular domain CD44, p-ERK1/2 and soluble CD44 in the ILF. Finally, MIF was found to be associated with soluble CD44 in the ILF. CONCLUSIONS: CD74 is present in the rat urothelium. SP increases CD74 and intracellular domain CD44 in the bladder, while stimulating the release of soluble CD44 and MIF into the ILF. MIF interacts with soluble CD44 in the ILF and it is available to bind with CD74 in the bladder to exert proinflammatory effects. Therefore, a mechanistic model is emerging to explain the proinflammatory effects of MIF in this acute model of bladder inflammation. Possible clinical implications are discussed.

Animals↗
PubMed1994

A sulfated proteoglycan as a novel ligand for CD44.

We have identified a novel ligand for CD44, a cell surface glycoprotein implicated in tumor metastasis and lymphocyte homing. When the mouse T cell line CTLL-2 was transfected with cDNA encoding a hemopoietic form of mouse CD44, CTLL-2 cells exhibited a new self-adhesive phenotype, forming large aggregates. The aggregation was blocked by neutralizing anti CD44 monoclonal antibody but unaffected by hyaluronidase, indicating the involvement of CD44 and its non-hyaluronate ligand in the cell aggregation. The ability to induce CD44-dependent aggregation was found in culture supernatants of CTLL-2 and its CD44 transfectants. The use of CD44-immunoglobulin chimeric protein revealed that CTLL-2 and its transfectants synthesized a large-molecular weight protein (gp600) which bound specifically to CD44. The gp600 was readily labeled with radioactive sulfate, and treatment of gp600 with chondroitinase ABC or ACII generated a lower molecular weight species (18-22 kDa), suggesting that gp600 consists of a small core protein with chondroitin sulfate glycosaminoglycan side chains. However, binding of CD44 to glycosaminoglycans such as chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate was undetectable, suggesting either that a novel chondroitin-type glycosaminoglycan is recognized by CD44 or that a particular configuration of the glycosaminoglycan is required for recognition by CD44.

Animals↗
PubMed1997

CD44 and the adhesion of neoplastic cells.

CD44 is a family of transmembrane glycoproteins that act mainly as a receptor for hyaluronan. It can also bind some other extracellular matrix ligands (chondroitin sulphate, heparan sulphate, fibronectin, serglycin, osteopontin) with lower affinity. CD44 is encoded by a single gene containing 20 exons, 10 of which (v1-v10) are variant exons inserted by alternative splicing. The standard, ubiquitously expressed isoform of CD44, does not contain sequences encoded by these variant exons. Numerous variant isoforms of CD44 containing different combinations of exons v1-v10 inserted into the extracellular domain can be expressed in proliferating epithelial cells and activated lymphocytes. CD44 plays a significant role in lymphocyte homing. Both alternative splicing and glycosylation influence receptor function of the molecule, usually reducing its affinity to hyaluronan. The cytoplasmic domain of CD44 communicates with the cytoskeleton via ankyrin and proteins belonging to the ezrin-moesin-radixin family. Relatively little is known about the intracellular events following interactions of CD44 with its ligands. Some variant isoforms, especially those containing sequences encoded by v6-v10, are overexpressed in both human and animal neoplasms. In a rat pancreatic adenocarcinoma model one of the variant CD44 isoforms was proved to be determinant in the metastatic process. For some human neoplasms (carcinomas of the digestive tract, non-Hodgkin's lymphomas, thyroid carcinomas, and others) correlations have been made between the particular pattern of CD44 variants produced by neoplastic cells and clinicopathological parameters of tumours, such as grade, stage, presence of metastases, and survival. In vitro studies indicate that modifications of CD44 expression result in different ligand recognition and influence cell motility, invasive properties, and metastatic potential of experimental tumours. Investigation of CD44 neoexpression can be useful both in early cancer diagnosis and in predicting tumour behaviour. It can also contribute to better understanding of molecular mechanisms leading to neoplastic transformation.

Alternative Splicing↗
PubMed2005

CD44 attenuates metastatic invasion during breast cancer progression.

Metastatic invasion is the primary cause of breast cancer mortality, and adhesion receptors, such as CD44, are believed to be critical in this process. Historically, primary breast tumor epithelium has been investigated in isolation from other tissue components, leading to the common interpretation that CD44 and its primary ligand, hyaluronan, promote invasion. Here, we provide in vivo evidence showing CD44 antagonism to breast cancer metastasis. In a mouse model of spontaneously metastasizing breast cancer (MMTV-PyV mT), we found that loss of CD44 promotes metastasis to the lung. Localization studies, in combination with a novel hyaluronan synthase-GFP transgenic mouse, show a restricted pattern of expression for CD44 and hyaluronan. Whereas CD44 is expressed in tumor epithelium, hyaluronan synthase expression is restricted to stromal-associated cells. This distinct CD44 and hyaluronan pattern of distribution suggests a role for epithelial-stromal interaction in CD44 function. To define the relevance of this spatial regulation, we developed an in vitro invasion assay to emulate invasion into the extracellular matrix. Invasion of CD44-positive tumor cells was inhibited in hyaluronan-containing matrices, whereas blocking CD44-hyaluronan association increased invasion. Collectively, these data show that during breast cancer progression, hyaluronan-CD44 dynamics occurring through epithelial-stromal interactions are protective against metastasis.

Animals↗
PubMed2001

Localization and roles of CD44, hyaluronic acid and osteopontin in IgA nephropathy.

An important function of CD44 is to act as a cellular receptor for hyaluronic acid and osteopontin. Cell-matrix interactions mediated by the CD44/hyaluronic acid receptor-ligand pair are involved in the regulation of leukocyte migration and activation. Osteopontin is a molecule associated with cell adhesion and migration and functions through binding to CD44. This study examined whether CD44, hyaluronic acid and osteopontin participate in the progression of IgA nephropathy. CD44 was expressed in mesangial cells, crescents, tubular cells and interstitial infiltrating cells in areas of tubulointerstitial injury. Hyaluronic acid was deposited in the capillary tuft of adhesion, crescents and the periglomerular area, and around damaged tubules. Osteopontin was expressed in tubular cells and interstitial infiltrating cells in areas of tubulointerstitial injury. The glomerular and interstitial deposition of hyaluronic acid correlated with the glomerular and interstitial expression of CD44. The interstitial expression of CD44 correlated with the interstitial expression of osteopontin. The expression of both CD44 and osteopontin in the interstitium correlated with the extent of tubulointerstitial damage. The expression of CD44 in the interstitium correlated with the severity of chronic glomerular lesions. The glomerular and interstitial CD44 and hyaluronic acid expression correlated with proteinuria, and interstitial CD44 and hyaluronic acid expression correlated with creatinine clearance rate. In summary, this study suggests that CD44 participates in the progression of IgA nephropathy by binding hyaluronic acid and osteopontin.

Adolescent↗
PubMed2004

Different type and localization of CD44 on surface membrane of regenerative renal tubular epithelial cells in vivo.

BACKGROUND: CD44 is a transmembrane glycoprotein comprising an extracellular domain, a transmembrane domain, and a cytoplasmic tail. Previous studies demonstrated that CD44 was generally restricted to lateral-basal plasma membrane (PM) of epithelial cells, whether it localized on apical PM in vivo has not been clarified. METHODS: In this study, we used a gentamicin-induced acute tubular necrosis (ATN) and spontaneous recovery model in rats and two distinct antibodies, an anti-rat distal extracellular domain (OX49) of standard CD44 (CD44-OX49) and an anti-rat CD44 cytoplasmic tail (CD44CPT), to survey the localization of CD44-OX49 and CD44CPT on the PM in renal tubular epithelial cells in different recovery stages after ATN with immunohistochemistry and immunoelectron-microscopic examinations. RESULTS: CD44-OX49 was localized not only on the lateral-basal PM in tubular epithelial cells, but also on the apical surface membrane in PCNA-positive newly regenerative tubular epithelial cells in early recovery stages after ATN. However, CD44CPT was only localized on the lateral-basal PM. The immunoelectron-microscopic results showed that CD44-OX49 localization was changed from the apical to lateral to basal surface membrane in renal tubular epithelial cells during the recovery process after ATN, finally disappearing from basal PM when normal polarized epithelial cells formed. CONCLUSIONS: These results suggest that there were two types of CD44 including CD44 without a cytoplasmic tail localizing on the apical surface membrane related to newly regenerative epithelial cells, and CD44 with a cytoplasmic tail localizing on the lateral-basal PM related to establishment of tubular epithelial cell polarity after ATN in vivo.

Animals↗
PubMed2001

The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.

Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD44 to atherosclerosis, we bred CD44-null mice to atherosclerosis-prone apoE-deficient mice. We found a 50-70% reduction in aortic lesions in CD44-null mice compared with CD44 heterozygote and wild-type littermates. We demonstrate that CD44 promotes the recruitment of macrophages to atherosclerotic lesions. Furthermore, we show that CD44 is required for phenotypic dedifferentiation of medial smooth muscle cells to the "synthetic" state as measured by expression of VCAM-1. Finally, we demonstrate that hyaluronan, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and that the low-molecular-weight proinflammatory forms of hyaluronan stimulate VCAM-1 expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of hyaluronan inhibit smooth muscle cell proliferation. We conclude that CD44 plays a critical role in the progression of atherosclerosis through multiple mechanisms.

Animals↗
PubMed2003

A role for CD44 in an antigen-induced murine model of pulmonary eosinophilia.

Previous studies established that IL-5-producing CD4(+) T cells play a pivotal role in allergic respiratory inflammation. It was also reported that CD4(+) T cells express higher levels of CD44 in the airway than in peripheral blood of patients with allergic respiratory diseases. We have used experimental pulmonary eosinophilia induced in mice by Ascaris suum (Asc) extract to investigate the role of CD44 in the development of allergic respiratory inflammation. Intraperitoneal administration of anti-CD44 mAb prevented both lymphocyte and eosinophil accumulation in the lung. Anti-CD44 mAb also blocked antigen-induced elevation of Th2 cytokines as well as chemokines (CCL11, CCL17) in bronchoalveolar lavage fluid (BALF). Treatment with anti-CD44 mAb inhibited the increased levels of hyaluronic acid (HA) and leukotriene concentrations in BALF that typically result from antigen challenge. Anti-CD44 mAb also blocked antigen-induced airway hyperresponsiveness. An anti-CD44 mAb (IM7) inhibited the HA-binding ability of splenocytes associated with decreased levels of CD44. Soluble CD44 levels in serum were increased in Asc-challenged IM7-treated mice, but not in KM201-treated mice, compared with Asc-challenged rat IgG-treated mice. Ab's that block CD44-HA binding reduced allergic respiratory inflammation by preventing lymphocyte and eosinophil accumulation in the lung. Thus, CD44 may be critical for development of allergic respiratory inflammation.

Animals↗
PubMed1999

CD44 expression marks the onset of keratinocyte stratification and mesenchymal maturation into fibrous dermis in fetal human skin.

The cell surface glycoprotein CD44 is involved in active cell movement, cancer metastasis, and morphogenesis. We studied its expression in fetal human skin using an antibody specific for CD44v3 and another that recognizes all CD44 forms. In embryonic and early fetal skin, only cells with dendritic morphology expressed CD44. The first keratinocyte expression of CD44 occurred in the basal cells on the eleventh week. Later, the suprabasal cells also turned positive, whereas periderm and the terminally differentiated cells remained negative at all stages. Maturation of the early mesenchyme towards dermis at the eleventh week was associated with an increase in the number of CD44-positive cells, and later the fibrous extracellular matrix also became CD44-positive. During hair induction, the epithelium showed a transient downregulation of CD44. Later, the follicular cells regained CD44. Cells in the primordial dermal papilla displayed a continuously strong signal. The sweat gland anlagen showed faint CD44 positivity. Exon 3 was present in the CD44 of keratinocytes and their derivatives but was absent in dermis. CD44 expression in human fetal skin is a relatively late event, associated with maturation and adult-type differentiation both in epidermal keratinocytes and in dermal fibroblastic cells.(J Histochem Cytochem 47:1617-1624, 1999)

Dermis↗
PubMed2002

CD44 variant-specific antibodies trigger hemopoiesis by selective release of cytokines from bone marrow macrophages.

Hemopoiesis is regulated by the complex interplay between the bone marrow microenvironment and hemopoietic stem cells and progenitors. The local production of cytokines plays a critical role in this process. Using long-term bone marrow cultures, we show here that monoclonal antibodies directed against the CD44 v4 and CD44 v6 epitopes stimulate myelopoiesis (CD44 v4 and CD44 v6) and lymphopoiesis (CD44 v6). In the bone marrow cell population, CD44 v4 and CD44 v6 epitopes are found virtually exclusively on double-positive bone marrow macrophages. The anti-CD44 v4 and v6 antibodies act on bone marrow macrophages to stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF) production (v4 and v6) and interleukin-6 (IL-6) production (v6). This profile of cytokine production explains the differential stimulation of hemopoiesis by the 2 antibodies. We suggest that the antibodies mimic ligand(s) that stimulate GM-CSF or IL-6 production by bone marrow-derived macrophages by binding to CD44 family members that bear CD44 v4 and CD44 v6 epitopes on these cells.

Amino Acid Sequence↗
PubMed2002

The role of CD44 in the development and prognosis of head and neck squamous cell carcinomas.

CD44, the product of a single gene, exists as several isoforms generated by alternative exon splicing and posttranslational modifications, and is widely distributed in different cells and tissues including those of squamocellular origin. CD44 is a cell surface glycoprotein involved in many cellular processes acting as a receptor for cell to cell or cell to matrix adhesion, as a signal transmitter and as a growth factor-presenting molecule. Numerous studies based on immunohistochemical analyses of paraffin-embedded or frozen tissue sections using different monoclonal antibodies to CD44 isoforms and molecular biological techniques have provided evidence that in many types of tumours there is overexpression of CD44 isoforms and aberrant processing of immature CD44 transcripts relative to non-neoplastic control tissues, suggesting a role of CD44 in tumour development and progression. In contrast to these malignancies, one or more of the CD44 splice-variant isoforms are down-regulated in squamous cell carcinomas of the head and neck. CD44-deficient mice develop normally without giving rise to spontaneous tumours, but CD44-negative cells appear to be more susceptible to oncogenic transformation. Reduction in the expression of CD44 may confer growth advantage and malignant properties to tumour cells. The clinical significance of CD44 in squamous cell carcinomas of the head and neck as a tumour marker for cancer diagnosis and prognosis is discussed.

Animals↗
PubMed2000

Cytokine regulation of CD44 expression on rat intestinal epithelial cells.

CD44 comprises a family of type I transmembrane glycoproteins that is expressed on a wide range of cells including those of epithelial, lymphoid and myeloid lineage. Although expression of CD44 in the small intestine is typically localised in the crypts of Lieberkuhn, we have reported the expression of CD44 on mature, intestinal villus epithelial cells during the development of small bowel allograft rejection. The mechanisms underlying CD44 up-regulation are unknown, although it may be influenced by localised cytokine production. This study used flow cytometry to assess the effects of recombinant IFN-gamma and TNF-alpha on CD44 expression and hyaluronan binding by the rat small intestinal epithelial cell lines, RIE and IEC 6. IFN-gamma upregulated CD44 expression on RIE (155% of unstimulated control) and IEC 6 (209% of unstimulated control) cells, whereas TNF-alpha had no effect. IFN-gamma had no qualitative effect on CD44, as binding of the ubiquitously expressed extracellular matrix polysaccharide hyaluronan was unchanged. RIE and IEC 6 cells expressed the 82 kDa and 130 kDa major isoforms of CD44, however cytokine stimulation did not affect the expression of these, nor did stimulation induce the expression of other variants. In summary, these findings demonstrate that CD44 expression by intestinal epithelial cells can be regulated by cytokines, yet their ability to bind hyaluronan and the isoform of the expressed CD44 remains unaltered. It appears that localised inflammatory conditions and cytokine production may modify epithelial cell expression of CD44, however the physiological role for such a response has yet to be elucidated.

Alternative Splicing↗
PubMed1998

Calcium- and calmodulin-dependent PMA-activation of the CD44 adhesion molecule.

The ability of the CD44 adhesion molecule to interact with its ligand hyaluronic acid (HA) is tightly regulated. CD44-positive mouse LB lymphoma cells are unable to bind HA unless activated by the tumor promoter phorbol 12-myristate 13-acetate (PMA). PMA causes a dose-dependent increase in both CD44 expression level and HA-binding capacity, with the binding of HA observed only above a threshold amount of CD44 molecules. This induction of HA-binding as well as the increase in CD44 expression are prevented by cycloheximide, suggesting a requirement for new additional CD44 molecules on the cell surface and/or cooperating proteins. In the present study, we have investigated which of the signal transduction pathways activated by PMA leads to the increased CD44 expression with subsequent acquisition of HA-binding capacity. By comparing the influence of each inhibitory agent on PMA-activated LB lymphoma cells versus that on a constitutive HA-binder cell line derived from LB cells (designated HA9 cells), we could distinguish between an effect on the PMA-activation phase and a one on the HA-binding phase. Our data show that the PMA-induced HA-binding could not be blocked by agents inhibiting protein kinase C (PKC) (staurosporine, sphingosine, polymyxin B, quercetin) or genestein, an inhibitor of tyrosine protein kinases. However, this PMA response was strongly inhibited by calmodulin antagonists (chlorpromazine, trifluoperazine, W-7) and the calcium blocker verapamil. The calmodulin antagonists inhibited the PMA-induced increase in CD44 expression on LB cells, but had no influence on the ability of the constitutive HA-binder HA9 cell line to interact with HA, indicating an effect on the PMA induction phase rather than on the binding itself. Verapamil also blocked the PMA-induced increase in CD44 expression on LB cells, but in addition it slightly reduced the ability of the HA9 cells to bind HA without affecting their CD44 expression level. In conclusion, our data suggest that CD44 activation by PMA is calcium and calmodulin dependent, rather than mediated by protein kinase C.

Animals↗
PubMed1999

Characterization of CD44 induction by IL-1: a critical role for Egr-1.

The adhesion molecule CD44 is a multifunctional, ubiquitously expressed glycoprotein that participates in the process of leukocyte recruitment to sites of inflammation and to their migration through lymphatic tissues. In this study, we have investigated the effect of the proinflammatory cytokine IL-1alpha on CD44 gene expression in the human immortalized endothelial cell line ECV304. Immunoblotting of cell extracts showed constitutive expression of a 85-kDa protein corresponding to the standard form of CD44, which was potently up-regulated following IL-1alpha treatment. Furthermore, IL-1alpha induced expression of v3- and v6-containing isoforms of CD44, which migrated at 110 and 140-180 kDa, respectively. The effect of IL-1alpha on CD44 standard, v3- and v6-containing isoforms was dose and time dependent and was inhibited in the presence of IL-1 receptor antagonist. To elucidate the molecular mechanisms regulating CD44 expression in response to IL-1alpha, we investigated the effect of IL-1alpha on CD44 mRNA expression. Reverse-transcriptase PCR and Northern analysis demonstrated an increase in CD44 mRNA expression indicating a transcriptional mechanism of control by IL-1alpha. Furthermore, IL-1alpha increased expression of a reporter gene under the control of the CD44 promoter (up to -1.75 kb). The effect of IL-1alpha was critically dependent on the site spanning -151 to -701 of the promoter. This effect required the presence of an Egr-1 motif at position -301 within the CD44 promoter since mutation of this site abolished responsiveness. IL-1alpha also induced Egr-1 expression in these cells. These studies therefore identify Egr-1 as a critical transcription factor involved in CD44 induction by IL-1alpha.

Cell Line, Transformed↗