The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin  1 and FAK staining. When cells were migrating, integrin  1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.
When cells are migrating, caveolin-1, the principal protein component of caveolae, is excluded from the leading edge and polarized at the cell rear. The dynamic feature depends on a specific sequence motif that directs intracellular trafficking of the protein. Deletion mutation analysis revealed a putative polarization domain at the N terminus of caveolin-1, between amino acids 32-60. Alanine substitution identified a minimal sequence of 10 residues ( 46 TKEIDLVNRD 55 ) necessary for caveolin-1 rear polarization. Interestingly, deletion of amino acids 1-60 did not prevent the polarization of caveolin-1 in human umbilical vein endothelial cells or wild-type mouse embryonic fibroblasts because of an interaction of Cav 61-178 mutant with endogenous caveolin-1. Surprisingly, expression of the depolarization mutant in caveolin-1 null cells dramatically impeded caveolae formation. Furthermore, knockdown of caveolae formation by methyl--cyclodextrin failed to prevent wild-type caveolin-1 rear polarization. Importantly, genetic depletion of caveolin-1 led to disoriented migration, which can be rescued by full-length caveolin-1 but not the depolarization mutant, indicating a role of caveolin-1 polarity in chemotaxis. Thus, we have identified a sequence motif that is essential for caveolin-1 rear polarization and caveolae formation.Caveolae are specific microdomains of the plasma membrane that were discovered more than 50 years ago (1). In endothelial cells, numerous vesicles appeared to derive from the uniformly flask-shaped invaginations, suggesting the endocytic potential of caveolae (2). Although the function of caveolae as transport vesicles mediating endocytosis and transcytosis remained obscure (3, 4), the identification, cloning, and characterization of caveolar coat proteins, caveolins, has increased our knowledge of caveolae, and a good body of evidence implicates caveolae in a specialized form of delivery of membrane components, extracellular ligands, bacterial toxins, and nonenveloped virus in several cell types (5-8). The caveolae-mediated endocytic pathway differs from that mediated by clathrincoated pits. It is sensitive to protein kinase C inhibitors and cholesterol depletion (by filipin), and in some cells it is involved in the activation of protein tyrosine kinases (9). Phosphorylation at tyrosine 14 of caveolin-1 (Cav-1) 2 may be required for the internalization of caveolae. The mechanism controlling caveolae trafficking remains unclear, but it apparently involves both microtubule and actin cytoskeletons (10). Surprisingly, using Cav-1 as a marker for caveolae, recent studies demonstrate that caveolae are rich in a variety of signaling molecules, with the implication that caveolae may function in the regulation of signal transduction. Given these views, an attractive hypothesis would be whether caveolae could carry signaling machinery to different locations of the cell to spatially organize signaling events. Indeed, Anderson and colleagues (11) have shown recently that concomitant with the relocation o...
Angiogenesis, the development of new blood vessels from preexisting capillary, is required for tumor growth and metastasis. The process is not fully understood yet, but involves endothelial cell proliferation, migration and differentiation. Recently, we have shown that overexpression of caveolin-1, a putative transformation suppressor gene, inhibits VEGFR-2 and MEK-1-mediated mitogenic signal to the nucleus. Conversely, angiogenic activators suppress caveolin-1 expression in endothelial cells. However, whether caveolin-1 expression affects endothelial cell proliferation is not clear. In the present study, we infect human endothelial cells with adenovirus expressing caveolin-1 and show that transient overexpression of caveolin-1 dramatically inhibits the proliferation of human endothelial cells. Consistent with caveolin-1 functioning as an inhibitor for protein kinases, overexpression of caveolin-1 inhibits the activity of VEGFR-2 (KDR) and down-stream p42/44 MAP kinase. Furthermore, overexpression of caveolin-1 prevents VEGF-induced down-regulation of the cyclin-dependent kinase inhibitor p27 kip1 and Rb phosphorylation, and subsequently arrests endothelial cells in the G 0 /G 1 phase. Thus, our results suggest that caveolin-1, as a negative regulator of endothelial cell proliferation, may be a potential target for the control of angiogenesis.
Caveolin-1 (Cav-1) plays an important role in the organization of signaling molecules involved in a variety of signaling pathways, including those mediating cell motility. Here we show that amino acids K47-K57 of Cav-1 are a highly conserved sequence in Cav-1 and Cav-3 proteins, and that expression of either K47-K57 deletion Cav-1 mutant or wild-type Cav-2 that lacks this sequence exhibits a non-polarized distribution pattern. Expression of K47-K57 in Cav-2 leads to Cav-2 polarity, suggesting that expression of K47-K57 is sufficient to direct caveolin polarity. Importantly, we show that expression of this sequence is both necessary and sufficient to promote cell directional migration. Thus, our results support the conclusion that Cav-1 polarity is critical for cell directional migration.
During tumorigenesis, a heterotypic interface exists between cancer and stromal cells that can both support and repress tumor growth. In the breast, studies have demonstrated a pro-tumorigenic role for adipocytes. However, the molecular mechanisms by which breast cancer cells coopt adipocytes remain elusive. Studying breast tumors and normal adjacent tissue (NAT) from several patient cohorts and mouse models, we show that lipolysis and lipolytic signaling are activated in NAT. We investigate the tumor-adipocyte interface and find that functional gap junctions form between breast cancer cells and adipocytes. As a result, cAMP, a critical lipolysis-inducing signaling molecule, is transferred from breast cancer cells to adipocytes and activates lipolysis in a gap junction-dependent manner; a fundamentally new mechanism of lipolysis activation in adipocytes. We find that gap junction formation depends upon connexin 31 (Cx31), and that Cx31 is essential for breast tumor growth and activation of lipolysis in vivo. Thus, direct tumor celladipocyte interaction is critical for tumorigenesis and may serve as a new therapeutic target in breast cancer.One sentence summary: Gap junctions between breast cancer cells and adipocytes transfer cAMP and activate lipolysis in the breast tumor microenvironment.Keywords: Breast cancer, triple-negative breast cancer, TNBC, adipocyte, gap junction, lipolysis, cAMP, connexin 31, Cx31, GJB3All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/277939 doi: bioRxiv preprint first posted online Mar. 7, 2018; Camarda R, et al. 2018 -preprint version -www.biorxiv.org 2 Materials and Methods:3CB imaging protocol The 3CB method combines dual-energy X-ray mammography attenuations and a breast thickness map to solve for the three unknowns: water, lipid, and protein content (1). We used Hologic Selenia full-field digital mammography system (Hologic, Inc.) to image women with 3CB. Two dual energy mammograms were acquired on each woman's affected breast using a single compression. The first exposure was made under conditions of regular clinical screening mammogram. The second mammogram was acquired at a fixed voltage (39 kVp) and mAs for all participants. A high energy exposure (39 kVp/Rh filter) was made using an additional 3-mm plate of aluminum in the beam to increase the average energy of the high energy image. We limited the total dose of this procedure to be approximately 110% of the mean-glandular dose of an average screening mammogram. The images were collected under an investigational review board approval to measure breast composition. The breast thickness map was modeled using the SXA phantom (2). The thickness validation procedure concluded in a weekly scanning of specially designed quality assurance phantom (3). The calibration standards and 3CB algorithms are described in full elsewhere...
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