Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie.

BACKGROUND: Prion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease. RESULTS: In the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR. CONCLUSIONS: The present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.

Nucleation and growth of integrin adhesions.

We present a model that provides a mechanistic understanding of the processes that govern the formation of the earliest integrin adhesions ex novo from an approximately planar plasma membrane. Using an analytic analysis of the free energy of a dynamically deformable membrane containing freely diffusing receptors molecules and long repeller molecules that inhibit integrins from binding with ligands on the extracellular matrix, we predict that a coalescence of polymerizing actin filaments can deform the membrane toward the extracellular matrix and facilitate integrin binding. Monte Carlo simulations of this system show that thermally induced membrane fluctuations can either zip-up and increase the radius of a nucleated adhesion or unzip and shrink an adhesion, but the fluctuations cannot bend the ventral membrane to nucleate an adhesion. To distinguish this integrin adhesion from more mature adhesions, we refer to this early adhesion as a nouveau adhesion.

Molecular genetic studies of a human epidermal autoantigen (the 180-kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and alpha 6 integrin.

The 180-kD bullous pemphigoid autoantigen (BP180) is a component of the hemidesmosome, a cell-matrix connector. This protein is oriented in a type II fashion in the membrane of the hemidesmosome and is a hybrid collagen (classified as type XVII). We have analyzed the fate of various mutant BP180 molecules transfected into several different cell types. A protein, D1, lacking the collagen-like extracellular domains of BP180 polarizes normally in 804G epithelial cells and colocalizes with other hemidesmosomal components in the plane of the basal cell surface. However, deletion of a stretch of 36 amino acids located at the NH2 terminus of D1 induces an apical polarization of the protein (D1-36N) in the cell surface of 804G cells. Deletion of the 27-amino acid noncollagenous extracellular domain that is located immediately after the membrane spanning domain of BP180 results in a failure of D1-27C protein to codistribute with other hemidesmosomal components despite its basal localization in transfected 804G cells. In FG cells, which lack their own BP180, transfected D1 protein localizes with the alpha 6 beta 4 integrin heterodimer. In HT1080 cells, which do not possess BP180 or beta 4 integrin, D1 protein localizes with alpha 6 beta 1 integrin while both the D1-27C and D1-36N proteins do not. Moreover, D1 protein coprecipitates with alpha 6 integrin from extracts of HT1080 transfectants. Taken together, these results suggest that the NH2-terminal domain of BP180 determines polarization of BP180 while the noncollagenous extracellular domain of BP180 stabilizes its interactions with other hemidesmosomal components, such as alpha 6 integrin. Perturbation of this latter domain by human bullous pemphigoid autoantibodies may explain the loss of epidermal cell-dermis attachment that characterizes the BP disease.

Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization.

Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.

Integrin beta 3 cytoplasmic tail is necessary and sufficient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrin-associated protein.

Using a K562 cell transfection model, we have previously described a novel relationship between the integrins alpha v beta 3 and alpha 5 beta 1. alpha v beta 3 ligation was able to inhibit alpha 5 beta 1-mediated phagocytosis without effect on alpha 5 beta 1-mediated adhesion. The alpha v beta 3-dependent inhibition apparently required a signal transduction cascade as it was reversed by inhibitors of serine/threonine kinases. Now, we have studied the mechanisms of signal transduction in this system and have found that the beta 3 cytoplasmic tail is both necessary and sufficient for initiation of the signal leading to inhibition of alpha 5 beta 1 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in alpha v beta 3 signal transduction, mimics the effects of alpha v beta 3 ligation only when the beta 3 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of alpha 5 beta 1, ligation of alpha v beta 3/IAP does not prevent acquisition of this high affinity state. We conclude that alpha v beta 3/IAP ligation initates a signal transduction cascade, dependent upon the beta 3 cytoplasmic tail, which inhibits the phagocytic function of alpha 5 beta 1 at a step subsequent to modulation of integrin affinity.

CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium.

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.

Molecular cloning of integrin-associated protein: an immunoglobulin family member with multiple membrane-spanning domains implicated in alpha v beta 3-dependent ligand binding.

Integrin Associated Protein (IAP) is a 50-kD membrane protein which copurifies with the integrin alpha v beta 3 from placenta and coimmunoprecipitates with beta 3 from platelets. IAP also is functionally associated with signal transduction from the Leukocyte Response Integrin. Using tryptic peptide sequence, human and murine IAP cDNAs have been isolated. The protein has an extracellular amino-terminal immunoglobulin domain that binds all monoclonal anti-IAP antibodies. The carboxy-terminal region is highly hydrophobic with three or five membrane-spanning segments and a short hydrophilic tail. Immunofluorescence microscopy suggests that this hydrophilic tail is located on the inside of the cytoplasmic membrane. Monoclonal anti-IAP antibody inhibits the binding of vitronectin-coated beads to alpha v beta 3 on human erythroleukemia cells, and polyclonal anti-IAP recognizes hamster IAP on CHO cells and inhibits vitronectin bead binding. When CHO cells are transfected with human IAP, monoclonal anti-human antibody completely inhibits vitronectin bead binding. These data suggest a model in which ligand binding by alpha v beta 3 is regulated by IAP.

Protein kinase C-dependent effects of 12(S)-HETE on endothelial cell vitronectin receptor and fibronectin receptor.

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid induced a nondestructive and reversible endothelial cell (EC) retraction. 12(S)-HETE induced EC retraction was inhibited by protein kinase C inhibitors calphostin C and staurosporine but not by the protein kinase A inhibitor H8. The role of EC integrins alpha v beta 3 and alpha 5 beta 1 in 12(S)-HETE induced EC retraction was investigated. In confluent EC cultures, alpha v beta 3 is localized to focal adhesions at both the cell body and cell-cell borders and is colocalized with vinculin-containing focal adhesions. In contrast, alpha 5 beta 1 is primarily enriched at the cell-cell borders, demonstrating codistribution with cell cortical microfilaments and extracellular fibronectin. Both receptors were functional in mediating cell-cell or cell-matrix interactions based on the observations that specific antibodies inhibited EC adhesion to intact subendothelial matrix and disrupted the monolayer integrity. 12(S)-HETE induced a multistep, temporally defined redistribution of the alpha v beta 3-containing focal adhesions, leading to an eventual decrease in alpha v beta 3 plaques in the retracted ECs. This effect of 12(S)-HETE was inhibited by calphostin C but not by H8. The alterations of alpha v beta 3-containing focal adhesions preceded the development of EC retraction. 12(S)-HETE also enhanced EC alpha v beta 3 surface expression as revealed by immunofluorescence, flow cytometry, and digitized image analysis. 12(S)-HETE-induced alpha v beta 3 rearrangement (i.e., decreased focal adhesion localization and enhanced surface expression) did not result from altered mRNA transcription (as revealed by semi-quantitative RT-PCR analysis) or protein translation (as revealed by Western blotting). In contrast to its effect on alpha v beta 3, 12(S)-HETE did not demonstrate a temporally related, well-defined effect on the distribution pattern and the surface expression of alpha 5 beta 1, although the cell-cell border staining pattern of alpha 5 beta 1 was disrupted due to EC retraction. It is concluded that 12(S)-HETE-induced decrease of alpha v beta 3 localization to focal adhesions may contribute to the development of EC retraction and that 12(S)-HETE induced increase in alpha v beta 3 surface expression may promote adhesion of inflammatory leukocytes as well as tumor cells to endothelium.

Association of insulin receptor substrate-1 with integrins.

Insulin stimulation was found to promote association of the alpha v beta 3 integrin (a vitronectin receptor) with insulin receptor substrate-1 (IRS-1), an intracellular protein that mediates insulin signaling by binding other signaling molecules, including growth factor receptor-bound protein 2 (Grb2) and phosphatidylinositol-3' kinase. Insulin-treated cells expressing the alpha v beta 3 integrin showed 2.5 times more DNA synthesis when plated on vitronectin than on other substrates, whereas cells expressing another vitronectin receptor, alpha v beta 5, did not show this difference. The association between integrin and IRS-1 may be a mechanism for the synergistic action of growth factor and extracellular matrix receptors.

Regulation of integrin function by the urokinase receptor.

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.

Analysis of CD36 binding domains: ligand specificity controlled by dephosphorylation of an ectodomain.

The protein CD36 is a membrane receptor for thrombospondin (TSP), malaria-infected erythrocytes, and collagen. Three functional sequences were identified within a single disulfide loop of CD36: one that mediates TSP binding (amino acids 87 to 99) and two that support malarial cytoadhesion (amino acids 8 to 21 and 97 to 110). One of these peptides (p87-99) is a consensus protein kinase C (PKC) phosphorylation site. Dephosphorylation of constitutively phosphorylated CD36 in resting platelets and a megakaryocytic cell line led to the loss of collagen adhesion and platelet reactivity to collagen, with a reciprocal increase in TSP binding. PKC-mediated phosphorylation of this ectodomain resulted in a loss of TSP binding and the reciprocal acquisition of collagen binding. In site-directed mutagenesis studies, when the threonine phosphorylation site was changed to alanine, CD36 was expressed in a dephosphorylated state and bound to TSP constitutively.

Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins.

Extracellular matrix (ECM) glycoproteins regulate neuronal development and axonal growth. In this paper, the ECM glycoprotein vitronectin was identified and localized in the embryonic chick neuroretina. To identify potentially important neurite outgrowth-promoting molecules, responses of embryonic chick retinal neurons to vitronectin and thrombospondin, another retinal ECM constituent, were examined. These neurons were shown to attach and extend neurites on either glycoprotein. Integrins containing the alpha v or beta 1 subunits mediate both responses to vitronectin and neurite outgrowth on thrombospondin. Attachment to thrombospondin was inhibited by heparin, suggesting that neurons also utilize a proteoglycan or sulfated glycolipid as a receptor for this glycoprotein. Thus, retinal neurons use specific receptors to interact with vitronectin and thrombospondin, two glycoproteins present in the embryonic neuroretina, suggesting roles for these ligands and their receptors in retinal development.

Smooth, rough and upside-down neocortical development.

Lissencephaly, which means 'smooth cortex', is caused by defective neuronal migration during development of the cerebral cortex and has devastating clinical consequences. 'Classical' lissencephaly seems to reflect mutations in regulators of the microtubule cytoskeleton, whereas 'cobblestone' lissencephaly is caused by mutations in genes needed for the integrity of the basal lamina of the central nervous system. Reelin, which is mutated in a third type of lissencephaly, may represent a unifying link because it encodes an extracellular protein that regulates neuronal migration and may also regulate the microtubule cytoskeleton.

Thrombospondin-platelet interactions. Role of divalent cations, wall shear rate, and platelet membrane glycoproteins.

The role of thrombospondin, a multifunctional matrix glycoprotein, in platelet adhesion is controversial: both adhesive and antiadhesive properties have been attributed to this molecule. Because shear flow has a significant influence on platelet adhesion, we have assessed thrombospondin-platelet interactions both under static and flow conditions. The capacity of thrombospondin to support platelet adhesion depended upon its conformation. In a Ca(2+)-depleted conformation, such as in citrated plasma, thrombospondin was nonadhesive or antiadhesive as it inhibited platelet adhesion to fibrinogen, fibronectin, laminin, and von Willebrand factor by 30-70%. In a Ca(2+)-replete conformation, however, thrombospondin effectively supported platelet adhesion. Shear rate influenced this adhesion; percent surface coverage on thrombospondin increased from 5.4 +/- 0.3 at 0 s-1 to 41.5 +/- 6.7 at 1,600 s-1. In contrast to the extensive platelet spreading observed on fibronectin at all shear rates, platelet spreading on thrombospondin occurred only sporadically and at high shear rates. GPIa-IIa, GPIIb-IIIa, GPIV, and the vitronectin receptor, which are all proposed platelet receptors for thrombospondin, were not solely responsible for platelet adhesion to thrombospondin. These results suggest that thrombospondin may play a dual role in adhesive processes in vivo: (a) it may function in conjunction with other adhesive proteins to maintain optimal platelet adhesion at various shear rates; and (b) it may serve as a modulator of cellular adhesive functions under specific microenvironmental conditions.

Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.

Sarcoglycan immunoreactivity is lacking in infantile hypertrophic pyloric stenosis. A confocal laser scanning microscopic study.

OBJECTIVES: The Dystrophin-Glycoprotein Complex (DGC) is a large multisubunit complex that plays a crucial role in maintaining the structural integrity and physiology of muscle fibers. Dystrophin has been reported to be absent in the pyloric muscle of infantile hypertrophic pyloric stenosis (IHPS) patients. The present study was designed to investigate the other two patterns of DGC (dystroglycan and sarcoglycan complexes) in normal pyloric muscle and their possible modifications in IHPS patients. METHODS: Ten pyloric muscle biopsies were obtained from babies operated for IHPS and five control pylorus biopsy taken at autopsy from cases without gastrointestinal disease. The DGC sub-complexes (beta-dystroglican and beta, delta- sarcoglycans) were localized immunohistochemically using specific monoclonal antibodies. The results were evaluated using a confocal laser scanning microscope. RESULTS: Positive immunolocalization of the two DGC sub complexes was demonstrated in the smooth muscle cells (SMCs) of the pyloric region of control patients. Similarly, a positive immune expression of beta-dystroglican was observed in the pyloric SMCs of IHPS patients. On the other hand a negative immunoreaction for sarcoglycans was recorded within the full thickness of the pyloric SMCs of these patients. CONCLUSIONS: The absence of sarcoglycans within the hypertrophied pyloric muscle may be a predisposing factor in the pathogenesis of IHPS since it could alter the normal physiology of SMCs through the modifications of structural integrity of sarcolemma and signaling between the extracellular and intracellular compartment.

The 72 kDa type IV collagenase is modulated via differential expression of alpha v beta 3 and alpha 5 beta 1 integrins during human melanoma cell invasion.

We have recently reported that concomitant with an increase in invasiveness, there is an increase in the expression and secretion of the matrix-degrading 72 kDa gelatinase A/type IV collagenase (MMP-2) in a moderately invasive human melanoma cell line (A375M) upon perturbation of the alpha v beta 3 classic vitronectin receptor. In the present study, we have extended these observations to include a highly invasive and metastatic melanoma cell line (C8161) which expresses a comparable amount of the alpha 5 beta 1 integrin (classic fibronectin receptor), but very little alpha v beta 3 integrin on its surface. When perturbed with an anti-alpha 5 beta 1 antibody, C8161 cells are 89% more invasive in vitro, and express and secrete increased levels of the gelatinase A. These changes were not elicited using antibodies to the alpha v beta 3 integrin. In addition, a 73% increase in invasion of C8161 cells through a fibronectin-enhanced matrix occurred, which could be abrogated by neutralizing antibodies to gelatinase A. Furthermore, we attempted to transiently mimic the invasive phenotype of the C8161 cells by diminishing the alpha v beta 3 integrin from the A375M cell surface through fluorescence-activated cell sorting selection or deoxynojirimycin treatment, and found these cells to be 30-50% more invasive than the parental population. These data suggest that alternative modulation and signaling events could be involved in melanoma tumor cell invasion as a result of the differential expression of integrins, and strictly cataloging the presence of these integrins is but an initial step in the analysis of their functional activity.

Adhesive interactions in angiogenesis and metastasis.

A variety of adhesive interactions must take place between the tumor cell and the host vasculature in order to potentiate both tumor expansion and metastatic tumor spread. The study of tumor cell and blood vessel adhesive interactions becomes essential for our understanding of the malignant process, especially with regard to organ-specific tumor metastasis. In this article we will review recent progress made in our understanding of the nature of (i) receptor mediated adhesion of endothelial cells to extracellular matrix components and (ii) adhesion of tumor cells to endothelial adhesion molecules and to components of the subendothelial basement membrane.

Cultured circulating mononuclear cells from osteopetrotic infants express the osteoclast-associated vitronectin receptor and form multinucleated cells in response to 1,25-dihydroxyvitamin D3.

Malignant osteopetrosis is characterized by impaired osteoclast activity. Osteoclasts derive from hematopoietic stem cells. In osteopetrosis, marrow cavities fail to develop, resulting in extramedullary hematopoiesis and the presence of stem cells in the bloodstream. Resistance to 1,25-(OH)2D3 may be involved in the pathogenesis of the disease. Sensitivity to 1,25-(OH)2D3, calcitonin sensitivity, and expression of the osteoclast-associated vitronectin receptor (VR) was examined in cultures of circulating mononuclear cells of seven osteopetrotic infants (1.5-6 months old). Since peripheral blood from age-matched children contains few stem cells, umbilical cord blood was used as control. Mononucleated cells were isolated by the Ficoll-Hypaque method and cultured (10(6) cells per ml) in alpha-MEM containing 20% horse serum in presence or absence of added 1,25-(OH)2D3. VR was identified by immunochemical staining with MAb 23C6. 1,25-(OH)2D3 at 10(-8) M significantly stimulated the formation of multinucleated cells (MNC) in cultures from all osteopetrotic patients and cord blood samples. Cells from three of five patients responded to 10(-9) M 1,25-(OH)2D3, the minimal stimulatory concentration for cord blood. Salmon calcitonin (100 ng/ml) partially inhibited the 10(-8) M 1,25-(OH)2D3-induced MNC formation in cultures from three of six patients and in cultures of all cord blood samples. In both types of cultures mononuclear cells and MNC cross-reacted with MAb 23C6, and 1,25-(OH)2D3 concentration did not influence the number and percentage of these cells. This study does not support the hypothesis of 1,25-(OH)2D3 resistance in osteopetrotic infants and shows that mononuclear cells expressing VR, possibly osteoclast progenitors, develop in cultures of circulating mononuclear cells from these infants. 1,25-(OH)2D3 may not be closely involved in VR expression.

Differentiation-controlled synthesis and binding of thrombospondin to granulosa cells.

Thrombospondin (TSP) is a large glycoprotein, synthesized by several matrix-forming cells and incorporated into their extracellular matrix. In several cell types its presence supports cell growth and proliferation. To investigate the role of this protein in cell differentiation, we studied the hormonal effect of TSP production and receptor-mediated binding to primary granulosa cells prepared from diethylstilbestrol-treated immature female rats. These cells can be induced to differentiate by FSH, 8-bromo-cAMP (8-Br-cAMP), or forskolin. Progesterone production is induced during differentiation, and its level of synthesis is an important manifestation of the differentiated phenotype. We find that undifferentiated granulosa cells synthesize and secrete TSP. The protein comprises about 0.5% of the total cell protein, and it is the major protein secreted in culture. Treatment of the cells with FSH or 8-Br-cAMP reduces TSP production dramatically, and forskolin completely inhibits it. In parallel, we observed that the undifferentiated cells bind TSP specifically with a Kd of 1.8 nM, and the number of binding sites per cell is 1.7 x 10(5). This binding can be prevented by excess TSP or an anti-TSP monoclonal antibody (B7-3). This ability to bind TSP is completely lost after induction of differentiation by FSH or 8-Br-cAMP. Our findings show that both the production and binding of TSP to granulosa cells are tightly controlled by normal cell differentiation and indicate that changes in TSP are correlated with the passage of the cell through the stages of maturation, a passage that also involves changes in cell shape and extracellular interactions and in the steroidogenic capacity of these cells.