Multifunctional Adhesion Molecule Research Articles

CD44 connects autophagy decline and ageing in the vascular endothelium

The decline of endothelial autophagy is closely related to vascular senescence and disease, although the molecular mechanisms connecting these outcomes in vascular endothelial cells (VECs) remain unclear. Here, we identify a crucial role for CD44, a multifunctional adhesion molecule, in controlling autophagy and ageing in VECs. The CD44 intercellular domain (CD44ICD) negatively regulates autophagy by reducing PIK3R4 and PIK3C3 levels and disrupting STAT3-dependent PtdIns3K complexes. CD44 and its homologue clec-31 are increased in ageing vascular endothelium and Caenorhabditis elegans, respectively, suggesting that an age-dependent increase in CD44 induces autophagy decline and ageing phenotypes. Accordingly, CD44 knockdown ameliorates age-associated phenotypes in VECs. The endothelium-specific CD44ICD knock-in mouse is shorter-lived, with VECs exhibiting obvious premature ageing characteristics associated with decreased basal autophagy. Autophagy activation suppresses the premature ageing of human and mouse VECs overexpressing CD44ICD, function conserved in the CD44 homologue clec-31 in C. elegans. Our work describes a mechanism coordinated by CD44 function bridging autophagy decline and ageing.

CD44s Assembles Hyaluronan Coat on Filopodia and Extracellular Vesicles and Induces Tumorigenicity of MKN74 Gastric Carcinoma Cells.

CD44 is a multifunctional adhesion molecule typically upregulated in malignant, inflamed and injured tissues. Due to its ability to bind multiple ligands present in the tumor microenvironment, it promotes multiple cellular functions related to tumorigenesis. Recent data has shown that CD44 and its principal ligand hyaluronan (HA) are carried by extracellular vesicles (EV) derived from stem and tumor cells, but the role of CD44 in EV shedding has not been studied so far. To answer this question, we utilized CD44-negative human gastric carcinoma cell line MKN74 manipulated to stably express CD44 standard form (CD44s). The effect of CD44s expression on HA metabolism, EV secretion, morphology and growth of these cells was studied. Interestingly, HAS2 and HYAL2 expression levels were significantly upregulated in CD44s-expressing cells. Cell-associated HA levels were significantly increased, while HA levels in the culture medium of CD44s-positive cells was lower compared to CD44s-negative MOCK cells. CD44s expression had no significant effect on the proliferation capacity of cells, but cells showed diminished contact inhibition. Superresolution imaging revealed that CD44s and HA were accumulated on filopodia and EVs secreted from CD44s-positive cells, but no differences in total numbers of secreted EV between CD44s-negative and -positive cells was detected. In 3D cultures, CD44s-expressing cells had an enhanced invasion capacity in BME gel and increased spheroidal growth when cultured in collagen I gel. No significant differences in mitotic activity, tumor size or morphology were detected in CAM assays. However, a significant increase in HA staining coverage was detected in CD44s-positive tumors. Interestingly, CD44s-positive EVs embedded in HA-rich matrix were detected in the stromal areas of tumors. The results indicate that CD44s expression significantly increases the HA binding capacity of gastric cancer cells, while the secreted HA is downregulated. CD44s is also carried by EVs secreted by CD44s-expressing cells. These findings highlight the potential usefulness of CD44s and its ligands as multipurpose EV biomarkers, because they are upregulated in inflammatory, injured, and cancer cells and accumulate on the surface of EVs secreted in these situations.

CD44 is a RAS/STAT5-regulated invasion receptor that triggers disease expansion in advanced mastocytosis

AbstractThe Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and invasion of neoplastic stem cells in various myeloid malignancies. Although mast cells (MCs) reportedly express CD44, little is known about the regulation and function of this receptor in neoplastic cells in systemic mastocytosis (SM). We found that clonal CD34+/CD38− stem cells, CD34+/CD38+ progenitor cells, and CD117++/CD34− MCs invariably express CD44 in patients with indolent SM (ISM), SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia (MCL). In addition, all human MCL-like cell lines examined (HMC-1, ROSA, and MCPV-1) displayed cytoplasmic and cell-surface CD44. We also found that expression of CD44 in neoplastic MCs depends on RAS-MEK and STAT5 signaling and increases with the aggressiveness of SM. Correspondingly, higher levels of soluble CD44 were measured in the sera of patients with advanced SM compared with ISM or cutaneous mastocytosis and were found to correlate with overall and progression-free survival. To investigate the functional role of CD44, a xenotransplantation model was employed using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and a short hairpin RNA (shRNA) against CD44. In this model, the shRNA-mediated knockdown of CD44 resulted in reduced MC expansion and tumor formation and prolonged survival in SCID mice compared with HMC-1.2 cells transduced with control shRNA. Together, our data show that CD44 is a RAS-MEK/STAT5-driven MC invasion receptor that correlates with the aggressiveness of SM. Whether CD44 can serve as therapeutic target in advanced SM remains to be determined in forthcoming studies.

Assay of serum CEACAM1 as a potential biomarker for breast cancer

BackgroundCarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a widely expressed multi-functional adhesion molecule reported to serve as a serum biomarker in several types of cancer. However, the serum CEACAM1 expression in breast cancer is unclear. We investigated the serum concentrations of CEACAM1 in patients with breast cancer and determine the potential of serum CEACAM1 as a breast cancer biomarker. MethodsSerum specimens were obtained from 33 patients with breast cancer, 30 patients with benign breast diseases and 34 healthy donors. The serum CEACAM1 concentrations were examined by an enzyme-linked immunosorbent assay (ELISA). ResultsThe serum CEACAM1 concentrations in the malignant group (532ng/ml) were significantly higher than those of the benign group (423ng/ml) and healthy control group (386ng/ml) (both p<0.001). Based on univariable logistic regression, serum CEACAM1 concentrations significantly predicted breast cancer versus normal controls or benign breast diseases. Area under receiver operating characteristic curve (ROC) for serum CEACAM1 was 0.925(95% CI: 0.866–0.984). The optimal cut-off concentration of CEACAM1 was 475.82ng/ml for discriminating breast cancer from normal controls. ConclusionSerum concentrations of CEACAM1 may serve as a useful indicator for the presence of breast cancer.

The neuroplastins: multifunctional neuronal adhesion molecules--involvement in behaviour and disease.

The neuroplastins np65 and np55 are neuronal and synapse-enriched immunoglobulin (Ig) superfamily cell adhesion molecules that contain 3 and 2 Ig domains, respectively. Np65 is neuron specific whereas np55 is expressed in many tissues. They are multifunctional proteins whose physiological roles are defined by the partner proteins they bind to and the signalling pathways they activate. The neuroplastins are implicated in activity-dependent long-term synaptic plasticity. Thus neuroplastin-specific antibodies and a recombinant peptide inhibit long-term potentiation in hippocampal neurones. This is mediated by activation of the p38MAP kinase signalling pathway, resulting in the downregulation of the surface expression of GluR1 receptors. Np65, but not np55, exhibits trans-homophilic binding. Both np65 and np55 induce neurite outgrowth and both activate the FGF receptor and associated downstream signalling pathways. Np65 binds to and colocalises with GABA(A) receptor subtypes and may play a role in anchoring them to specific synaptic and extrasynaptic sites. Most recently the neuroplastins have been shown to chaperone and support the monocarboxylate transporter MCT2 in transporting lactate across the neuronal plasma membrane. Thus the neuroplastins are multifunctional adhesion molecules which support neurite outgrowth, modulate long-term activity-dependent synaptic plasticity, regulate surface expression of GluR1 receptors, modulate GABA(A) receptor localisation, and play a key role in delivery of monocarboxylate energy substrates both to the synapse and to extrasynaptic sites. The diverse functions and range of signalling pathways activated by the neuroplastins suggest that they are important in modulating behaviour and in relation to human disease.

Vitronectin increases vascular permeability by promoting VE-cadherin internalization at cell junctions.

BackgroundCross-talk between integrins and cadherins regulates cell function. We tested the hypothesis that vitronectin (VN), a multi-functional adhesion molecule present in the extracellular matrix and plasma, regulates vascular permeability via effects on VE-cadherin, a critical regulator of endothelial cell (EC) adhesion.Methodology/Principal FindingsAddition of multimeric VN (mult VN) significantly increased VE-cadherin internalization in human umbilical vein EC (HUVEC) monolayers. This effect was blocked by the anti-αVβ3 antibody, pharmacological inhibition and knockdown of Src kinase. In contrast to mult VN, monomeric VN did not trigger VE-cadherin internalization. In a modified Miles assay, VN deficiency impaired vascular endothelial growth factor-induced permeability. Furthermore, ischemia-induced enhancement of vascular permeability, expressed as the ratio of FITC-dextran leakage from the circulation into the ischemic and non-ischemic hindlimb muscle, was significantly greater in the WT mice than in the Vn −/− mice. Similarly, ischemia-mediated macrophage infiltration was significantly reduced in the Vn −/− mice vs. the WT controls. We evaluated changes in the multimerization of VN in ischemic tissue in a mouse hindlimb ischemia model. VN plays a previously unrecognized role in regulating endothelial permeability via conformational- and integrin-dependent effects on VE-cadherin trafficking.Conclusion/SignificanceThese results have important implications for the regulation of endothelial function and angiogenesis by VN under normal and pathological conditions.

Dermatopontin Promotes Epidermal Keratinocyte Adhesion via α3β1 Integrin and a Proteoglycan Receptor

Dermatopontin, an extracellular matrix component initially purified from bovine dermis, promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited by both EDTA and heparin and was mediated in part by alpha3beta1 integrin. A synthetic peptide (DP-4, PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dose-dependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence (GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide affinity column. These results suggest that HaCaT cells adhere to dermatopontin through alpha3beta1 integrin and a heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin plays a role as a multifunctional adhesion molecule for epidermal cells.

The Role of Multifunctional Adhesion Molecules in Spermatogenesis and Sperm Function: Lessons from Hemostasis and Defense?

The functional maintenance of the vascular endothelial cell barrier depends on different homo- and heterotypic adhesion systems involving tight junctions, junctional adhesion proteins, and cadherins. Upon inflammatory responses, vessel wall-dependent adhesion and transmigration of leukocytes involves the subtle orchestration of intercellular adhesion receptors and their counter-ligands on each cell type. Following tissue injury, the hemostatic/wound-healing process relies on various cell-associated adhesion receptors (particularly integrins) on platelets and vascular cells as well as on extracellular matrix (ECM) proteins to warrant sealing of the wound. In particular, integrin-binding ECM adhesion molecules mediate firm anchorage as well as cellular motility in cooperation with pericellular proteolytic systems. Accumulating evidence indicates that such cell-anchored and ECM adhesion proteins, which are crucial in vascular defense processes, are also expressed in the testicular epithelium and in gametes to mediate the timely events of spermatid movement during spermatogenesis in the testis and to contribute to the various phases of the fertilization process, culminating in sperm-oocyte fusion, respectively. We explore the multifunctional roles of junctional adhesion molecules, nectins, integrins, ECM proteins, and others beyond their role in defense and hemostasis as important contributors in spermatogenesis and sperm function.

Localization of CD44 (Hyaluronan Receptor) and Hyaluronan in Rat Mandibular Condyle

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.

Targeting leukocyte integrins in human diseases

As our understanding of integrins as multifunctional adhesion and signaling molecules has grown, so has their recognition as potential therapeutic targets in human diseases. Leukocyte integrins are of particular interest in this regard, as they are key molecules in immune-mediated and inflammatory processes and are thus critically involved in diverse clinical disorders, ranging from asthma to atherosclerosis. Antagonists that interfere with integrin-dependent leukocyte trafficking and/or post-trafficking events have shown efficacy in multiple preclinical models, but these have not always predicted success in subsequent clinical trials (e.g., ischemia-reperfusion disorders and transplantation). However, recent successes of integrin antagonists in psoriasis, inflammatory bowel disease, and multiple sclerosis demonstrate the tremendous potential of antiadhesion therapy directed at leukocyte integrins. This article will review the role of the leukocyte integrins in the inflammatory process, approaches to targeting leukocyte integrins and their ligands, and the results of completed clinical trials.

Selectins—an emerging target for drug delivery

Selectins are multifunctional adhesion molecules that mediate the initial interactions between circulating leukocytes and cells of the endothelium. First identified over a decade ago, selectins have provided insight into areas as diverse as normal lymphocyte homing, leukocyte recruitment during inflammatory responses, carbohydrate ligand biosynthesis and adhesion-mediated signalling. Of late, selectins were introduced as targets for drug delivery in the development of new anti-inflammatory therapeutics and in anti-cancer therapy. This review will examine the selectins and their ligands with a focus on recent findings on their role in physiology and pathophysiology as well as the emerging role of selectins as targets in controlled drug delivery.

The effect of 3-aminobenzamide, inhibitor of poly(ADP-ribose) polymerase, on human osteosarcoma cells

This study demonstrates that in human osteosarcoma cells treatment with 3-aminobenzamide (3-AB), a potent inhibitor of poly(ADP-ribose) polymerase (PARP), induces morphological and biochemical features of differentiation, the duration of which depends on whether or not the normal RB gene is expressed. In Saos-2 cells expressing a non-functional Rb protein, 3-AB treatment induced the formation of transient, short dendritic-like protrusions. In RB-transfected-Saos-2 cells (a clone previously generated in our laboratory that shows stable expression of wild-type Rb protein), 3-AB induced marked and prolonged changes with the formation of long dendritic-like protrusions and the appearance of stellate (osteocyte-like) cells. In MG-63 cells producing a wild-type Rb protein, 3-AB treatment had more marked effects, with a larger number of cells assuming the stellate appearance of osteocytes, which were connected to each other via junctions resembling small channels. Regardless of cell type, at some point after 3-AB treatment the differentiative attempt failed and the cells died. Death was apoptotic, as demonstrated by chromatin condensation and fragmentation, specific cleavage of PARP and Lamin-B, processing of caspase-3 and the appearance of Bax immunoreactive species. Enzymatic assay and RT-PCR of alkaline phosphatase (ALP) - an enzyme whose levels markedly decrease when osteoblasts undergo terminal differentiation into osteocytes - showed that 3-AB treatment markedly lowered ALP expression. Simultaneously, 3-AB treatment markedly increased the expression of CD44, a transmembrane multifunctional adhesion molecule and sensitive marker of osteocytic differentiation. This study hypothesizes a cross-talk between pRb and PARP and suggests that PARP may be a useful target for anticancer drugs.

Targeting dendritic cells with CD44 monoclonal antibodies selectively inhibits the proliferation of naive CD4+ T-helper cells by induction of FAS-independent T-cell apoptosis.

CD44 is a multifunctional adhesion molecule that has been shown to be a costimulatory factor for T-cell activation in vitro and in vivo. The aim of the present study was to expand these findings by characterizing the role of CD44 during dendritic cell (DC) antigen presentation to naive, resting T cells. Certain monoclonal antibodies (mAbs) directed against all CD44 isoforms (pan CD44), or against the epitope encoded by the alternatively spliced exon v4 (CD44v4), dose-dependently inhibited the capacity of murine DC to induce proliferation of naive alloreactive T cells. Preincubation of the T cells or DC with these CD44 mAbs revealed that the effect was dependent upon mAb binding to DC, but not to T cells. DC treated with anti-pan CD44 and anti-CD44v4 mAbs induced CD4+ T-cell apoptosis, as shown by annexin V staining and TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assays. However, CD4+ T-cell apoptosis was not dependent on the Fas/Fas ligand (Fas/FasL) system, as DC from FasL-deficient (Gld) mice and T cells from Fas-deficient (Lpr) mice were still susceptible to apoptosis induced by CD44-treated DC. To investigate whether CD44 treatment of DC affects early T-cell/DC interactions, time-lapse video microscopy was performed using peptide-specific T cells from T-cell receptor (TCR) transgenic mice. Interestingly, calcium signalling in CD4+ T cells was significantly diminished following interaction with CD44 mAb-treated DC, but this was not observed in CD8+ T cells. Taken together, we found that perturbation of distinct epitopes of CD44 on DC interfere with early Ca2+ signalling events during the activation of CD4+ T cells, resulting in T-cell apoptosis.

Immunohistochemical Study for CD44v6 in Hepatocellular Carcinoma and Cholangiocarcinoma

CD44 is a multifunctional adhesion molecule in cell-to-cell and cell-to-matrix interactions. This transmembrane glycoprotein exists in either standard or variant form, with the variation originating in alternative splicing. This study was designed to evaluate the role of CD44v6, one of the CD44 isoforms, in hepatocellular carcinoma and cholangiocarcinoma. MATERIALS AND METGODS: Immunohistochemical expression of CD44v6 was studied in 7 normal livers, 14 hepatocellular carcinomas and 16 cholangiocarcinomas, that were formalin fixed and paraffin embedded. CD44v6 was frequently expressed in the normal hepatocytes and hepatocellular carcinomas. Expression was not noted in the normal bile duct within the portal tract. CD44v6 was positively expressed in some of the proliferating bile ducts (43%) and cholangiocarcinomas (69%). CD44v6 expression may be more important in the stepwise carcinogenesis of the bile duct than in the normal hepatocyte, but further study is needed.

Selectins: critical mediators of leukocyte recruitment

Selectins are multi-functional adhesion molecules that mediate the initial interactions between circulating leukocytes and the endothelium. First identified over a decade ago, selectins have provided insight into areas as diverse as normal lymphocyte homing, leukocyte recruitment during inflammatory responses, carbohydrate ligand biosynthesis and adhesion-mediated signalling. This review will examine the selectins and their ligands with a focus on recent findings using knockout technology as well as the emerging role of selectins as signalling molecules.

Spatial and temporal distribution of CD44 and osteopontin in fracture callus.

The multifunctional adhesion molecule CD44 is a major cell-surface receptor for hyaluronic acid (HUA). Recent data suggest that it may also bind the ubiquitous bone-matrix protein, osteopontin (OPN). Because OPN has been shown to be a potentially important protein in bone remodelling, we investigated the hypothesis that OPN interactions with the CD44 receptor on bone cells participate in the regulation of the healing of fractures. We examined the spatial and temporal patterns of expression of OPN and CD44 in healing fractures of rat femora by in situ hybridisation and immunohistochemistry. We also localised HUA in the fracture callus using biotinylated HUA-binding protein. OPN was expressed in remodelling areas of the hard callus and was found in osteocytes, osteoclasts and osteoprogenitor cells, but not in cuboidal osteoblasts which were otherwise shown to express osteocalcin. The OPN signal in osteocytes was not uniformly distributed, but was restricted to specific regions near sites where OPN mRNA-positive osteoclasts were attached to bone surfaces. In the remodelling callus, intense immunostaining for CD44 was detected in osteocyte lacunae, along canaliculi, and on the basolateral plasma membrane of osteoclasts, but not in the cuboidal osteoblasts. HUA staining was detected in fibrous tissues but little was observed in areas of hard callus where bone remodelling was progressing. Our findings suggest that OPN, rather than HUA, is the major ligand for CD44 on bone cells in the remodelling phase of healing of fractures. They also raise the possibility that such interactions may be involved in the communication of osteocytes with each other and with osteoclasts on bone surfaces. The interactions between CD44 and OPN may have important clinical implications in the repair of skeletal tissues.

Spatial and temporal distribution of CD44 and osteopontin in fracture callus

The multifunctional adhesion molecule CD44 is a major cell-surface receptor for hyaluronic acid (HUA). Recent data suggest that it may also bind the ubiquitous bone-matrix protein, osteopontin (OPN). Because OPN has been shown to be a potentially important protein in bone remodelling, we investigated the hypothesis that OPN interactions with the CD44 receptor on bone cells participate in the regulation of the healing of fractures. We examined the spatial and temporal patterns of expression of OPN and CD44 in healing fractures of rat femora by in situ hybridisation and immunohistochemistry. We also localised HUA in the fracture callus using biotinylated HUA-binding protein. OPN was expressed in remodelling areas of the hard callus and was found in osteocytes, osteoclasts and osteoprogenitor cells, but not in cuboidal osteoblasts which were otherwise shown to express osteocalcin. The OPN signal in osteocytes was not uniformly distributed, but was restricted to specific regions near sites where OPN mRNA-positive osteoclasts were attached to bone surfaces. In the remodelling callus, intense immunostaining for CD44 was detected in osteocyte lacunae, along canaliculi, and on the basolateral plasma membrane of osteoclasts, but not in the cuboidal osteoblasts. HUA staining was detected in fibrous tissues but little was observed in areas of hard callus where bone remodelling was progressing. Our findings suggest that OPN, rather than HUA, is the major ligand for CD44 on bone cells in the remodelling phase of healing of fractures. They also raise the possibility that such interactions may be involved in the communication of osteocytes with each other and with osteoclasts on bone surfaces. The interactions between CD44 and OPN may have important clinical implications in the repair of skeletal tissues.

CD44 is not an adhesive receptor for osteopontin.

Osteopontin is a secreted glycoprotein with adhesive and migratory functions. Cellular interactions with osteopontin are mediated through integrin receptors which recognize the RGD domain. Recently, CD44, a non-integrin, multifunctional adhesion molecule was identified as an osteopontin receptor. CD44 is a ubiquitous surface molecule that exists as a number of different isoforms, generated by alternative splicing. To analyze which forms of CD44 mediate binding to osteopontin, we used the standard form of CD44 as CD44-human immunoglobulin fusion proteins and several splice variants in enzyme-linked immunosorbant assays. Multiple preparations of osteopontin were used including native osteopontin derived from smooth muscle cells, human urinary osteopontin, full-length recombinant osteopontin, and two recombinant osteopontin fragments expected to be formed following thrombin cleavage. Our data show that although the CD44-hlg fusion proteins could interact with hyaluronic acid as expected, there was no interaction between CD44H, CD44E, CD44v3,v8-v10, or CD44v3 with osteopontin. These studies suggest that CD44-osteopontin interactions may not be common in vivo and may be limited to a specific CD44 isoform(s), and/or a particular modified form of osteopontin.

In vitro expression of soluble and cell surface-associated CD44 on endometrial cells from women with and without endometriosis.

Endometriosis is one of the most common benign gynaecological diseases, and attachment of retrogradely shed viable endometrial cells is considered to be important in its development. CD44 is a multifunctional adhesion molecule that undergoes alternative splicing, giving rise to different isoforms. The expression of cell surface-associated CD44 std, v4, v5, v6 and v10 variants before and after cytokine treatment was investigated in endometrial cultures derived from 10 endometriosis patients and 22 women without the disease using immunocytochemistry. The immunoreactivity of soluble CD44 std, v5 and v6 variants was measured in culture medium using an enzyme immunoassay kit. We report on the presence of soluble CD44 in endometrial culture supernatants. In particular, circulating CD44 standard form levels were significantly higher than levels of splice variants. We also found that both epithelial and stromal cells express surface-associated CD44 molecules in a distinct pattern and that this expression is not modulated by tumour necrosis factor (TNF)-alpha or/and interleukin 1 (IL-1) alpha/beta. Finally, cell surface-associated as well as soluble CD44 expression was similar in the two groups. Our results indicate that endometrial cells can serve as a source of circulating CD44, but a direct role in the pathogenesis of endometriosis is rather improbable.

Regulation of CD44 Gene Expression by the Proinflammatory Cytokine Interleukin-1β in Vascular Smooth Muscle Cells

The CD44 gene codes for a family of alternatively spliced, multifunctional adhesion molecules that participate in extracellular matrix binding, lymphocyte activation, cell migration, and tumor metastasis. In a mouse model of transplant-associated arteriosclerosis, CD44 protein was induced in the neointima of allografted vessels and colocalized with a subset of proliferating vascular smooth muscle cells (SMC). To elucidate the molecular mechanisms regulating CD44 expression in this model, we investigated the regulation of CD44 gene expression by interleukin (IL)-1beta. Treatment of rat aortic SMC with IL-1beta resulted in a 5.3-fold increase in cell surface CD44 expression. Northern analysis showed that IL-1beta promoted a dose- and time-dependent induction of CD44 mRNA which reached 6.6-fold after 48 h, and nuclear run-on analysis showed that IL-1beta increased the rate of CD44 gene transcription within 8 h of stimulation. In transient reporter gene transfection experiments in rat aortic SMC, a 1.4-kilobase fragment of the mouse CD44 5'-flanking sequence mediated this response to IL-1beta. Regulation of CD44 gene expression by the proinflammatory cytokine IL-1beta may contribute to SMC phenotypic modulation in the pathogenesis of arteriosclerosis.