Primary human keratinocytes externalize stratifin protein via exosomes

Although, stratifin (SFN) is externalized by keratinocytes and stimulates the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts, its mechanism of externalization is not known. Here, we hypothesize that keratinocytes have a capacity to release stratifin through externalization of exosomes. To test this hypothesis, exosomes were purified from human keratinocyte conditioned medium (KCM) and analyzed for the presence of SFN by Western blot analysis using lysosomal-associated membrane protein 2 (LAMP-2) and heat shock cognate 70 (hsc70) as exosomal markers. The results showed the presence of SFN in keratinocyte lysate, concentrated KCM and exosomes, but not in concentrated unconditioned medium. Transmission electron microscopic examination revealed the presence of unique "saucer-like" structures characteristic of exosomes whose diameters were <100 nm. Similar to the recombinant SFN, the exosomes associated proteins stimulated MMP-1 expression in fibroblasts. Depletion of the exosomes markedly reduced this MMP-1 stimulatory effect. To further statistically confirm these findings, fibroblasts were treated with three different exosome preparations and the finding showed more than 7.4-fold increase in the level of MMP-1 in the treated cells. Furthermore, we found that approximately 1% of the total proteins contained in exosomes correspond to SFN. In conclusion, this study is the first report showing that keratinocytes have the capacity to produce exosomes through which some intracellular proteins such as SFN, with MMP-1 stimulating activity for fibroblasts, is externalized into keratinocyte microenvironment.     

Dermal fibroblasts influence the expression profile of 14-3-3 proteins in human keratinocytes

We have previously demonstrated that the release of some of the 14-3-3 isoforms from keratinocytes is able to influence the expression of key matrix metalloproteinases (MMPs) in dermal fibroblasts. Conversely, in this study we aimed to investigate whether dermal fibroblasts possess the ability to modulate the expression of 14-3-3 proteins in keratinocytes. In order to address this question, human keratinocytes and dermal fibroblasts were harvested and co-cultured. Intra- and extracellular levels of 14-3-3 proteins (β, η, γ, and σ) were analyzed using western blot analysis, and the gene expression was further assessed by quantitative real-time polymerase chain reaction. Gene analysis revealed an up-regulation of all four 14-3-3 isoforms of interest. In addition, the findings of this study reveal a significant increase in the intracellular levels of 14-3-3 γ and σ in keratinocytes co-cultured with fibroblasts compared to those of the mono-cultured control keratinocytes. Mechanistic investigations also demonstrated the capacity of several mitogen-activated protein kinase-specific inhibitors to markedly reduce induction of 14-3-3 σ in keratinocytes stimulated with fibroblast-conditioned medium. The study concluded that dermal fibroblasts possess the ability to influence the expression of several 14-3-3 isoforms (notably γ and σ) in keratinocytes, suggesting that the two cell types might be capable of bi-directionally influencing the protein expression of one another in vivo.     

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication.     

Stimulating the Release of Exosomes Increases the Intercellular Transfer of Prions

Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP^C, into the disease-associated isoform, PrP^Sc. The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP^C, leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.     

Identification of different isoforms of 14-3-3 protein family in human dermal and epidermal layers

We have previously demonstrated a high level of stratifin, also known as 14-3-3 sigma in differentiated keratinocyte cell lysate and conditioned medium (CM). In this study, we asked the question of whether other 14-3-3 isoforms are expressed in human dermal fibroblasts, keratinocytes, intact dermal and epidermal layers of skin. In order to address this question, total proteins extracted from cultured cells or skin layers were subjected to western blot analysis using seven different primary antibodies specific to well-known mammalian isoforms, beta, gamma, epsilon, eta, sigma, tau, and zeta of 14-3-3 protein family. The autoradiograms corresponding to each isoform were then quantified and compared. The results revealed the presence of very high levels of all seven isoforms in cultured keratinocyte and conditioned medium. With the exception of tau isoform, other 14-3-3 isoforms were also present in intact epidermal layer of normal skin. The profile of 14-3-3 proteins in whole skin was similar to that of epidermis. In contrast, only gamma 14-3-3 isoform, was present in dermal layer obtained from the same skin sample. On the other hand, cultured fibroblasts express a high level of beta, epsilon, gamma and eta and a low level of zeta and tau, but not sigma isoform. However, the levels of 14-3-3 epsilon, gamma and eta were barely detectable in fibroblast conditioned medium. Further, we also used immunohistochemical staining to identify the 14-3-3 isoform expressing cells in human skin sections. The finding revealed different expression profile for each of these isoforms mainly in differentiated keratinocytes located within the layer of lucidum. However, fibroblasts located within the dermal layer did not show any detectable levels of these proteins. In conclusion, all members of 14-3-3 proteins are expressed by cells of epidermal but not dermal layer of skins and that these proteins are mainly expressed by differentiated keratinocytes.     

Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.     

Dynamics of exosome internalization and trafficking

Cells release exosomes into extracellular medium. Although the important roles of exosomes in many physiological and pathological processes are being revealed, the mechanism of exosome–cell interaction remains unclear. In this article, employing real‐time fluorescence microscopy, the motion of exosomes on the plasma membrane or in the cytoplasm of recipient PC12 cells was observed directly. In addition, several motion modes of exosomes were revealed by single particle tracking (SPT). The changes between motion modes were also detected, presenting the dynamic courses of exosome attachment onto plasma membrane and exosome uptake. Octadecyl rhodamine B chloride (R18) was found to be useful to distinguish endocytosis from fusion during exosome uptake. Colocalization with organelle markers showed exosomes were sorted to acidic vesicles after internalization. The results provide new sight into the exosome–cell interaction mode and the intercellular trafficking of exosomes. This study will help to understand the roles of exosomes at cell level. J. Cell. Physiol. 228: 1487–1495, 2013. © 2012 Wiley Periodicals, Inc.     

Tumour‐derived exosomal miR‐3473b promotes lung tumour cell intrapulmonary colonization by activating the nuclear factor‐κB of local fibroblasts

Tumour‐derived exosomes have been shown to induce pre‐metastatic niche formation, favoring metastatic colonization of tumour cells, but the underlying molecular mechanism is still not fully understood. In this study, we showed that exosomes derived from the LLC cells could indeed significantly enhance their intrapulmonary colonization. Circulating LLC‐derived exosomes were mainly engulfed by lung fibroblasts and led to the NF‐κB signalling activation. Further studies indicated that the exosomal miR‐3473b was responsible for that by hindering the NFKB inhibitor delta's (NFKBID) function. Blocking miR‐3473b could reverse the exosome‐mediated NF‐κB activation of fibroblasts and decrease intrapulmonary colonization of lung tumour cells. Together, this study demonstrated that the miR‐3473b in exosomes could mediate the interaction of lung tumour cells and local fibroblasts in metastatic sites and, therefore, enhance the metastasis of lung tumour cells.     

Inhibiting Extracellular Vesicle Release from Human Cardiosphere Derived Cells with Lentiviral Knockdown of nSMase2 Differentially Effects Proliferation and Apoptosis in Cardiomyocytes,Fibroblasts and Endothelial Cells In Vitro

Numerous studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes may contribute to improved cardiac function. However, it has not yet been demonstrated by a genetic approach that exosome release contributes to the therapeutic effect of transplanted CDCs. By employing a lentiviral knockdown (KD) strategy against neutral spingomyelinase 2 (nSMase2), a crucial gene in exosome secretion, we have defined the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblast, endothelial cell and primary cardiomyocyte proliferation, cell death, migration and angiogenesis using a series of in vitro coculture assays. We found that secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 lentiviral KD and shRNAi expression was stable and constitutive. hCDC exosome release contributed to the angiogenic and pro-migratory effects of hCDCs on HUVECs, decreased proliferation of fibroblasts, and decreased apoptosis of cardiomyocytes. These in vitro reactions support a role for exosome secretion as a paracrine mechanism of stem cell-mediated cardiac repair in vivo. Importantly, we have established a novel tool to test constitutive inhibition of exosome secretion in stem cell populations in animal models of cardiac disease.     

Differential Calcium Requirements For Growth of Mouse Skin Epithelial and Fibroblast Cells

Concentrations of extracellular Ca⁺⁺ optimum for growth of cell types of mesodermal origin have been reported to be up to 100-fold higher than concentrations optimal for epidermal or other epithelial lining cells. In order to examine Ca⁺⁺ requirements of epithelial v. fibroblastic cells derived from a common tissue source, prior to prolonged culture, freshly isolated mouse epidermal keratinocytes, hair follicle cells and dermal fibroblasts were plated at high density or at clonal density in medium ranging from 0.014 to 1.4 mM Ca⁺⁺. Epithelial skin cells grew best at Ca⁺⁺ levels below 0.1 mM while dermal fibroblasts grew best at a Ca⁺⁺ concentration of 1.4 mM. the epithelial cell types exhibited marked morphologic changes in response to Ca⁺⁺, while the fibroblasts did not. These results suggest that the variations in Ca⁺⁺ response between lining epithelium and mesenchymal cells resulted from inherent differences in these cell types, but a mechanism for such differential effects has not yet been defined.     

Cellular Internalization of Exosomes Occurs Through Phagocytosis

Exosomes play important roles in many physiological and pathological processes. However, the exosome–cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non‐phagocytic cells. Most exosomes were observed attached to the plasma membrane of non‐phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate‐modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3‐kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant‐negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin‐coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome–cell interactions and the exosome intracellular trafficking pathway.     

α11β1 integrin‐mediated MMP‐13‐dependent collagen lattice contraction by fibroblasts: Evidence for integrin‐coordinated collagen proteolysis

We have previously determined that integrin α11β1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11^?/? mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11^?/? mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11‐deficient iPDL cells, including matrix metalloproteinase‐13 (MMP‐13) and cathepsin K. α11^?/? iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP‐13 protein expression levels. The MMP‐13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin‐mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11β1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11β1‐dependent matrix remodeling, involving MMP‐13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Tumor-derived exosomal miR-3157-3p promotes angiogenesis,vascular permeability and metastasis by targeting TIMP/KLF2 in non-small cell lung cancer

Metastasis is the main cause of death in patients with advanced lung cancer. The exosomes released by cancer cells create tumor microenvironment, and then accelerate tumor metastasis. Cancer-derived exosomes are considered to be the main driving force for metastasis niche formation at foreign sites, but the mechanism in Non-small cell lung carcinoma (NSCLC) is unclear. In metastatic NSCLC patients, the expression level of miR-3157-3p in circulating exosomes was significantly higher than that of non-metastatic NSCLC patients. Here, we found that miR-3157-3p can be transferred from NSCLC cells to vascular endothelial cells through exosomes. Our work indicates that exosome miR-3157-3p is involved in the formation of pre-metastatic niche formation before tumor metastasis and may be used as a blood-based biomarker for NSCLC metastasis. Exosome miR-3157-3p has regulated the expression of VEGF/MMP2/MMP9 and occludin in endothelial cells by targeting TIMP/KLF2, thereby promoted angiogenesis and increased vascular permeability. In addition, exosome miR-3157-3p promoted the metastasis of NSCLC in vivo.Subject terms: Cancer microenvironment, Non-small-cell lung cancer     

PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes

Exosomes are small vesicles secreted by different immune cells and which display anti-tumoral properties. Stimulation of RBL-2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.     

Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts

Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca²⁺] in cells loaded with the fluorescent Ca²⁺ probe Fura-2. Tryptase produced transient cytosolic Ca²⁺ mobilization in keratinocytes, but not in fibroblasts. Ca²⁺ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase-induced Ca²⁺ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR-1 and -3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR-2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca²⁺ mobilization, nor did it affect Ca²⁺ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR-1 may be susceptible to proteases with chymotrypsin-like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells. J. Cell. Physiol. 176:365–373, 1998. © 1998 Wiley-Liss, Inc.     

Exosomes Derived from the Human Primary Colorectal Cancer Cell Line SW480 Orchestrate Fibroblast‐Led Cancer Invasion

In localized tumors, basement membrane (BM) prevents invasive outgrowth of tumor cells into surrounding tissues. When carcinomas become invasive, cancer cells either degrade BM or reprogram stromal fibroblasts to breach BM barrier and lead invasion of cancer cells into surrounding tissues in a process called fibroblast‐led invasion. However, tumor‐derived factors orchestrating fibroblast‐led invasion remain poorly understood. Here it is shown that although early‐stage primary colorectal adenocarcinoma (SW480) cells are themselves unable to invade Matrigel matrix, they secrete exosomes that reprogram normal fibroblasts to acquire de novo capacity to invade matrix and lead invasion of SW480 cells. Strikingly, cancer cells follow leading fibroblasts as collective epithelial‐clusters, thereby circumventing need for epithelial to mesenchymal transition, a key event associated with invasion. Moreover, acquisition of pro‐invasive phenotype by fibroblasts treated with SW480‐derived exosomes relied on exosome‐mediated MAPK pathway activation. Mass spectrometry‐based protein profiling reveals that cancer exosomes upregulate fibroblasts proteins implicated in focal adhesion (ITGA2/A6/AV, ITGB1/B4/B5, EGFR, CRK), regulators of actin cytoskeleton (RAC1, ARF1, ARPC3, CYFIP1, NCKAP1, ICAM1, ERM complex), and signalling pathways (MAPK, Rap1, RAC1, Ras) important in pro‐invasive remodeling of extracellular matrix. Blocking tumor exosome‐mediated signaling to fibroblasts therefore represents an attractive therapeutic strategy in restraining tumors by perturbing stroma‐driven invasive outgrowth.     

Adiponectin is partially associated with exosomes in mouse serum

Exosomes are membrane vesicles 30–120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo.     

Physical exosome:exosome interactions

Exosomes are extracellular nanovesicles that mediate a number of cellular processes, including intracellular signalling. There are many published examples of exosome–exosome dimers; however, their relevance has not been explored. Here, we propose that cells release exosomes to physically interact with incoming exosomes, forming dimers that we hypothesize attenuate incoming exosome‐mediated signalling. We discuss experiments to test this hypothesis and potential relevance in health and disease.