Exosomes: novel regulators of bone remodelling and potential therapeutic agents for orthodontics.

Recent studies suggest that exosomes are involved in intercellular communication required for the maintenance of healthy bone. Exosomes are small (30-150 nm in diameter) extracellular vesicles that are formed in multivesicular bodies and are released from cells as the multivesicular bodies fuse with the plasma membrane. Regulatory exosomes have the capacity to exert profound control over target cells. They can stimulate plasma membrane receptors and are also internalized by the target cell delivering proteins, lipids, small molecules and functional RNAs from the cell of origin. We and others have recently reported on regulatory exosomes from osteoclasts and osteoblasts. Key candidate molecules identified in exosome-based regulation of bone remodelling include receptor activator of nuclear factor kappa B (RANK), RANK-ligand (RANKL), ephrinA2, semaphorin 4D, microRNA-146a and microRNA- 214-3p. Exosomes will likely prove to be crucial elements in the communication networks integrating bone cells (osteoclasts, osteoblasts, osteocytes) and linking bone to other tissue. Exosomes collected from bone cells grown in culture may prove useful to augment bone remodelling associated with orthodontic force application or required for the repair of craniofacial bone. Various technologies allow exosomes to be engineered to improve their targeting and efficacy for therapeutic purposes. In summary, exosomes have emerged as important elements of the machinery for intercellular communication between bone cells. They hold great promise as therapeutic targets, biomarkers and therapeutic agents for orthodontists.

The use of cell culture platforms to identify novel markers of bone and dentin resorption.

OBJECTIVES: 1) To test the hypothesis that there would be proteomic differences in the composition of exosomes isolated from osteoclasts and odontoclasts and 2) to determine the clinical usefulness of these in vitro biomarker candidates. MATERIALS AND METHODS: Mouse bone marrow-derived precursors were cultured on either dentin or bone slices and allowed to mature and begin resorption. Exosomes were isolated from cell culture media and characterized by mass spectrometry. The proteomic data obtained from this in vitro study were compared with the data obtained from human samples in our previous work. RESULTS: There was a difference in the proteomic composition of exosomes from osteoclasts and odontoclasts. A total of 40 exosomal proteins were only present in osteoclast media, whereas six unique exosomal proteins were identified in odontoclast supernatants. Approximately 50% of exosomal proteins released by clastic cells in vitro can be found in oral fluids. CONCLUSION: Our data suggest that the mineralized matrix type plays a role in the final phenotypic characteristics of mouse clastic cells. Many in vitro biomarker candidates of bone and dentin resorption can also be found in human oral fluids, thus indicating that this approach may be a viable alternative in biomarker discovery.

Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation.

Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin (ß3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p<0.05) reduced the expression of NFATc1, CathK, OSCAR, FcRγ, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p<0.05) decreased CathK, OSCAR, FcRγ, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the calcineurin-NFAT signalling cascade to suppress key mediators of the ITAM pathway during late stage osteoclast differentiation and this is associated with a reduction in both osteoclast differentiation and activity.

The role of cell-substrate interaction in regulating osteoclast activation: potential implications in targeting bone loss in rheumatoid arthritis.

Analysis of tissues retrieved from the bone-pannus interface from patients with rheumatoid arthritis (RA) and studies in animal models of inflammatory arthritis provide strong evidence that osteoclasts, the cells that are essential for physiological bone resorption, are responsible for articular bone destruction in RA. However, current treatments that specifically target osteoclast-mediated bone resorption in RA have not been successful in preventing bone erosions, and new therapeutic strategies are needed. It has been noted that, although osteoclast precursors are present within the bone microenvironment at sites of pathological bone resorption, cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface and adjacent calcified cartilage. These findings provide evidence that, in addition to requirements for specific cytokines, interaction of osteoclast precursors with these mineralised matrices results in activation of specific signal pathways and the induction of unique gene products that are essential for terminal osteoclast differentiation and activation. These studies are designed to define the gene products and signalling pathways regulated by bone and calcified cartilage, to identify new molecular targets and novel therapeutic approaches for preventing osteoclast-mediated joint destruction in RA and related forms of pathological bone loss.

A role for the alphavbeta3 integrin in the transmigration of monocytes.

The beta2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin alphavbeta3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking beta3 integrins revealed weak migratory ability, whereas monocytes expressing beta3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains alphaL, beta2, alphav, or IAP, a protein functionally associated with alphavbeta3 integrin. Transfection of beta3 integrin chain cDNA into monocytes lacking beta3 integrins resulted in expression of the alphavbeta3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in alphaLbeta2-dependent locomotion on recombinant ICAM-1 which was enhanced by alphavbeta3 integrin occupancy. Antibodies against IAP were able to revert this alphavbeta3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of alphavbeta3 integrin could decrease monocyte binding to ICAM-1. In conclusion, we show that alphavbeta3 integrin modulates alphaLbeta2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival.

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.

A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function.

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.

Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts.

Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.

Role of cell-matrix interactions in osteoclast differentiation.

Osteoclast and their mononuclear cell precursors are present within the bone microenvironment at sites of physiologic and pathologic bone resorption. Analysis of tissues from sites of bone resorption reveal that cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface. We hypothesize that in addition to cytokines, components of the bone matrix and specific cell surface receptors on osteoclasts and their precursors play an essential role in determining the genetic profile and functional properties of fully differentiated resorbing osteoclasts. We have employed expression profiling, with an in vitro model of matrix-dependent osteoclast differentiation, to identify the molecular pathways by which bone matrix-interactions induce terminal osteoclast differentiation and activation. In preliminary studies, we have identified unique genes and transcriptional pathways that are induced by interaction of osteoclast precursors with specific components of the mineralized bone matrix. The authenticity of the gene profiles, as markers of osteoclast differentiation and activation, have been provisionally validated using an in vivo animal bone implantation model and by examination of tissues from patients with specific forms of pathologic osteoclast-mediated bone resorption. The ultimate goal of our studies is to identify new molecular targets for inhibiting osteoclast-mediated bone loss in disorders of pathologic bone loss. The early work of Walker et al. (Walker 1972) in parabiotic animals, and the subsequent studies of Burger et al. (Burger, Van der Meer, van de Gevel, et al. 1982) using a co-culture model with fetal bone rudiments and bone marrow-derived cells, have helped to establish that osteoclasts are derived from macrophage precursors of colony forming unit-macrophage (CFU-M lineage). As such, they share a common hematopoietic origin with other CFU-M lineage cells, including tissue macrophages that populate the lung (alveolar macrophages), liver (Kupfer cells), synovium (synovial macrophages) and other organs. They also share a common lineage

Characterization of Regulatory Extracellular Vesicles from Osteoclasts.

Extracellular vesicles (EVs), which include exosomes and ectosomes/microvesicles, have emerged as important intercellular regulators. EVs can interact with surface receptors of target cells and can transport luminal components, including messenger RNAs (mRNAs), microRNAs, and enzymes, to the cytosol of the target cell. Here, we show that hematopoietic cells grown in culture shed exosome-like EVs as they differentiate from preosteoclasts into osteoclasts. These EVs were between 25 and 120 nm (mean, 40 nm) in diameter determined by transmission electron microscopy. The exosome-associated markers CD63 and EpCAM were enriched in the isolated EVs while markers of Golgi and endoplasmic reticulum were not detected. Treatment of isolated hematopoietic cells with EVs did not affect their receptor activator of nuclear factor κB-ligand (RANKL)-stimulated differentiation into osteoclasts. However, EVs from osteoclast precursors promoted 1,25-dihydroxyvitamin D3-dependent osteoclast formation in whole mouse marrow cultures, and EVs from osteoclast-enriched cultures inhibited osteoclastogenesis in the same cultures. These data suggested that osteoclast-derived EVs are paracrine regulators of osteoclastogenesis. EVs from mature osteoclasts contained receptor activator of nuclear factor κB (RANK). Immunogold labeling showed RANK was enriched in 1 in every 32 EVs isolated from osteoclast-enriched cultures. Depletion of RANK-rich EVs relieved the ability of osteoclast-derived EVs to inhibit osteoclast formation in 1,25-dihydroxyvitamin D3-stimulated marrow cultures. In summary, we show for the first time that EVs released by osteoclasts are novel regulators of osteoclastogenesis. Our data suggest that RANK in EVs may be mechanistically linked to the inhibition of osteoclast formation. RANK present in EVs may function by competitively inhibiting the stimulation of RANK on osteoclast surfaces by RANKL similar to osteoprotegerin. RANK-rich EVs may also take advantage of the RANK/RANKL interaction to target RANK-rich EVs to RANKL-bearing cells for the delivery of other regulatory molecules.

Caveolae in human and murine osteoblasts.

Caveolae are 50- to 100-nm plasmalemmal vesicles formed by oligomerized caveolin, a 22-kDa phosphoprotein. These organelles have been implicated in critical signal transduction and molecular transport processes. Here, we show for the first time that osteoblasts express caveolin and have abundant caveolae. Membrane fractionation techniques indicate that osteoblast caveolin is found in detergent-resistant membranes that have the buoyant density characteristic of caveolae, whereas immunoblotting and reverse-transcription polymerase chain reaction (RT-PCR) show that osteoblasts express both caveolin-1 and -2 isoforms. Electron microscopy (EM) and immunofluorescence reveal the hallmarks of caveolae in osteoblasts: abundant 50- to 100-nm noncoated cell surface invaginations (caveolae) and abundant punctate clusters of immunostained caveolin.

A highly repeated retropseudogene-like sequence in DNA of the redbreasted merganser (Mergus serrator).

Two highly repeated nucleotide sequences (RBMI and RBMII) cloned from an EcoRI digest of DNA of the redbreasted merganser (Mergus serrator) account for approx. 5 to 10% of the DNA of M. serrator and the closely related Mergus merganser. Complete DNA digestion of seven members of the Mergini with EcoRI produces distinct, relatively species-specific patterns of a few high-Mr (greater than 1.5 kb) fragments of RBMI-like material. In such digests RBMII forms ladder-type patterns with monomers of approx. 200 bp. The sequence of a cloned 2.6-kb RBMI fragment from M. serrator contains several extended (up to 70 bp) and modified poly(dA) sequences, two open reading frames in opposite orientation to the longest poly(dA) sequence and two direct 10-bp repeats suggesting that RBMI is a rearranged retropseudogene-like element.

Mortality among the residents of the Three Mile Island accident area: 1979-1992.

The largest U.S. population exposed to low-level radioactivity released by an accident at a nuclear power plant is composed of residents near the Three Mile Island (TMI) Plant on 28 March 1979. This paper (a collaboration of The University of Pittsburgh and the Pennsylvania Department of Health) reports on the mortality experience of the 32,135 members in this cohort for 1979-1992. We analyzed standardized mortality ratios (SMRs) using a local comparison population and performed relative risk regression modeling to assess overall mortality and specific cancer risks by confounding factors and radiation-related exposure variables. Total mortality was significantly elevated for both men and women (SMRs = 109 and 118, respectively). All heart disease accounted for 43.3% of total deaths and demonstrated elevated SMRs for heart disease of 113 and 130 for men and women, respectively; however, when controlling for confounders and natural background radiation, these elevations in heart disease were no longer evident. Overall cancer mortality was similar in this cohort as compared to the local population (male SMR = 100; female SMR = 101). In the relative risk modeling, there was a significant effect for all lymphatic and hematopoietic tissue in males in relation to natural background exposure (p = 0.04). However, no trend was noted. We found a significant linear trend for female breast cancer risk in relation to increasing levels of TMI-related likely [gamma]-exposure (p = 0.02). Although such a relationship has been noted in other investigations, emissions from the TMI incident were significantly lower than in other documented studies. Therefore, it is unlikely that this observed increase is related to radiation exposure on the day of the accident. The mortality surveillance of this cohort does not provide consistent evidence that radioactivity released during the TMI accident has a significant impact on the mortality experience of this cohort to date. However, continued follow-up of these individuals will provide a more comprehensive description of the morbidity and mortality experience of the cohort.

Serum testosterone levels decrease in middle age in women with the polycystic ovary syndrome.

OBJECTIVE: To determine whether testosterone levels change as women with the polycystic ovary syndrome (PCOS) grow older. DESIGN: A follow-up cross-sectional study of a cohort of women with PCOS identified up to 20-25 years ago. SETTING: Women with PCOS were recruited primarily from practice records between 1970 and 1990. Voter registration tapes and household directories were used to identify age-, race-, and neighborhood-matched controls. PARTICIPANT(S): Eighty-four women with PCOS, 20-57 years of age, and 37 age-matched controls participating in a study of the risk for cardiovascular disease in women with PCOS. INTERVENTION(S): Clinical data were collected by questionnaire and fasting blood samples were obtained randomly throughout the menstrual cycle. MAIN OUTCOME MEASURE(S): Total and non-SHBG-bound testosterone levels. RESULT(S): Total and non-SHBG-bound testosterone levels were similar in women with PCOS who were 20-42 years of age but were reduced by approximately 50% among women 42-47 years of age and remained stable in women older than 47 years of age. Testosterone levels were increased in younger and older women with PCOS compared with controls but were similar to controls in women 42-47 years of age. CONCLUSION(S): Hyperandrogenism partly resolves before menopause in women with PCOS. This change may explain the tendency of women with PCOS to cycle regularly as they grow older. Testosterone levels remain elevated in older women with PCOS, however, and may contribute to their increased risk for cardiovascular disease, endometrial cancer, and other diseases.

Do polycystic-appearing ovaries affect the risk of cardiovascular disease among women with polycystic ovary syndrome?

OBJECTIVE: To determine if polycystic-appearing ovaries (PAO) are associated with differences in risk factors for cardiovascular disease among women with polycystic ovary syndrome (PCOS). DESIGN: Case-control sub-study. SETTING: Division of Reproductive Endocrinology, Magee-Womens Hospital. PATIENT(S): Women with PCOS (n = 63) and non-PCOS controls (n = 56). INTERVENTION: Transvaginal ultrasonography and single sample venipuncture. MAIN OUTCOME MEASURE(S): Ultrasound ovarian appearance, fasting insulin, lipoproteins, androgens, LH/FSH ratio, anthropomorphic measurements, and blood pressure. RESULT(S): Women with PCOS had higher androgen and fasting insulin levels, a more adverse lipid profile, greater waist-hip and LH/FSH ratios, and a larger ovarian volume than controls. Thirty-three percent of the cases with PCOS, but only 5% of controls, showed PAO on ultrasound study (P<.01). PCOS cases with and without PAO had comparable levels of fasting insulin, lipids, and blood pressures. PCOS cases with PAO had a higher LH/FSH ratio (P=.028), increased levels of serum androstenedione (P=.029) and testosterone (P=.055), and greater ovarian volume (P=.024) compared to non-PAO patients. CONCLUSION: Women with PCOS have greater cardiovascular risk than controls. Within PCOS cases, however, the ultrasound appearance of polycystic ovaries does not appear to further intensify the cardiovascular disease risk profile of these women.

Evidence for a dose-response relationship between occupational noise and blood pressure.

In this study, we investigated the role of occupational noise exposure and blood pressure among workers at 2 plants. A noise-exposed plant (plant 1, > or = 89 dBA) and a less-noise-exposed plant (plant 2, < or = 83 dBA) were chosen. Exposure was based on department-wide average noise measures; on the basis of job location and adjusting for layoffs during their employment at the plant, a cumulative time-weighted average noise level was calculated for each worker. The study population comprised 329 males in plant 1 and 314 males in plant 2. Their ages ranged from 40 to 63 y (mean ages = 49.6 and 48.7, respectively), and they had worked at least 15 y at the plant. The clinical examination was administered prior to the workday and measured height, weight, pulse, and blood pressure. In addition, we noted medical and personal-habits histories, including alcohol intake and cigarette smoking patterns. We used a questionnaire to determine in-depth occupation, military history, noisy hobbies, and family history of hypertension. When individuals who took blood-pressure medication were removed from the analysis, t tests for differences in average blood pressure between plants showed a mean systolic blood pressure of 123.3 mm Hg in plant 1 versus 120.8 mm Hg in plant 2 (p = .06) and a mean diastolic blood pressure of 80.3 mm Hg versus 77.8 mm Hg in Plant 1 and 2, respectively (p = .014). On the basis of data from the combined plants, multivariate analysis revealed that age, body mass index, cumulative noise exposure, current use of blood pressure medications, and alcohol intake were significant predictors for systolic blood pressure. Cumulative noise exposure was a significant predictor of diastolic blood pressure in plant 1 but not in plant 2, possibly reflecting a threshold effect.

Cloning and characterization of the murine beta(3) integrin gene promoter: identification of an interleukin-4 responsive element and regulation by STAT-6.

Expression of the alpha(v)beta(3) integrin by murine bone marrow macrophages is regulated by cytokines such as IL-4 and GM-CSF through transcriptional activation of the beta(3) subunit gene. To characterize the molecular mechanisms by which such regulation occurs, we isolated the murine beta(3) integrin promoter. To this end, we first cloned a full length beta(3) cDNA and used the 5'UTR and leader peptide coding sequence to identify genomic clones containing the beta(3) promoter region. The transcriptional start site, identified by primer extension and S1 nuclease assay, is 34 nt upstream of the translation initiation codon. A 1.1 kb fragment of the promoter region drives IL-4 responsive transcription in transiently transfected murine bone marrow macrophages. Deletion analysis of the beta(3) promoter indicates the IL-4 responsive element lies between -465 to -678 nt relative to the transcriptional start site. This promoter fragment contains two overlapping STAT consensus recognition sites and nuclear extracts from BMMs contain an IL-4-inducible DNA binding factor, identified by super shift analysis, as STAT-6. Furthermore, an oligonucleotide which includes the two STAT recognition sites residing in the IL-4 responsive region of the beta(3) promoter, competes for STAT-6 binding. Confirming IL-4 induction of the integrin subunit is specifically mediated by STAT-6, beta(3) mRNA is not enhanced in BMMs derived from STAT-6 deleted mice, which however, retain their capacity to respond to GM-CSF.

Characterization of a major tandemly repeated DNA sequence (RBMII) prevalent among many species of waterfowl (Anatidae).

We have investigated the evolution of a 190 base pair tandemly repeated DNA sequence (RBMII) in 27 different species of waterfowl. In this paper we show that the RBMII sequence is present in many species belonging to 7 of the 11 Anatid tribes. Inter- and intra-tribal differences in repeat presence indicate that, although the RBMII sequence has been maintained among widely divergent species, it is rapidly evolving. Restriction enzyme analyses suggest very different hierarchical repeat organizations among different species. DNA sequence comparisons of 32 cloned monomer units from five different species revealed what appears to be a nonrandom distribution of sequence divergence, as well as large differences (up to 25-fold) in intraspecific sequence variation between relatively closely related species.

Differential expression of alphav integrins in K1735 melanoma cells.

Tumor cell adherence to and migration on the extracellular matrix is an important aspect of cancer progression. This interaction with the extracellular matrix is mediated primarily through the integrin class of cell adhesion molecules. We identified a restricted expression of alphavbeta3 in highly metastatic K1735M2 and of alphavbeta5 in poorly metastatic K1735C23 murine melanoma cells. The highly metastatic cells were ten times more motile on vitronectin and fibronectin and approximately three times more invasive through a reconstituted basement membrane than the poorly metastatic cells. This motility was inhibited by addition of anti-beta3 antibodies. Injection of the alphavbeta3-negative K1735C23 cells into syngeneic mice resulted in the generation of a metastatic variant (K1735C23PM) that neo expressed the alphavbeta3 complex, indicating that expression of alphavbeta3 is required for K1735 melanoma metastasis. Injection of highly metastatic K1735M2 cells in the presence of blocking antibody to beta3 reduced tumor size by approximately 80%. Treatment of the K1735M2 cells with a retroviral antisense beta3 construct significantly reduced their expression of alphavbeta3 and also reduced their motility on extracellular matrix ligands and their invasion through a reconstituted basement membrane. In contrast, when the K1735C23 cells were treated with a construct containing the full-length beta3 cDNA, their motility on extracellular matrix proteins and invasion of a reconstituted basement membrane were significantly increased. These results indicate that alphavbeta3 is required for migration and invasion of K1735 melanoma cells in vitro and primary tumor growth and metastasis in vivo.