The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.

In different systems, cyclic adenosine monophosphate (cAMP) either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (TSH). The cAMP-dependent mitogenic pathway is unique as it is independent of mitogen-activated protein kinase activation and differs from growth factor–dependent pathways at the level of the expression of several protooncogenes/transcription factors. This study examined the involvement of D-type G1 cyclins and their associated cyclin-dependent kinase (cdk4) in the cAMP-dependent G1 phase progression of dog thyroid cells. Unlike epidermal growth factor (EGF)+serum and other cAMP-independent mitogens, TSH did not induce the accumulation of cyclins D1 and D2 and partially inhibited the basal expression of the most abundant cyclin D3. However, TSH stimulation enhanced the nuclear detection of cyclin D3. This effect correlated with G1 and S phase progression. It was found to reflect both the unmasking of an epitope of cyclin D3 close to its domain of interaction with cdk4, and the nuclear translocation of cyclin D3. TSH and EGF+serum also induced a previously undescribed nuclear translocation of cdk4, the assembly of precipitable cyclin D3–cdk4 complexes, and the Rb kinase activity of these complexes. Previously, cdk4 activity was found to be required in the cAMP-dependent mitogenic pathway of dog thyrocytes, as in growth factor pathways. Here, microinjections of a cyclin D3 antibody showed that cyclin D3 is essential in the TSH/ cAMP-dependent mitogenesis, but not in the pathway of growth factors that induce cyclins D1 and D2. The present study (a) provides the first example in a normal cell of a stimulation of G1 phase progression occurring independently of an enhanced accumulation of cyclins D, (b) identifies the activation of cyclin D3 and cdk4 through their enhanced assembly and/or nuclear translocation, as first convergence steps of the parallel cAMP-dependent and growth factor mitogenic pathways, and (c) strongly suggests that this new mechanism is essential in the cAMP-dependent mitogenesis, which provides the first direct demonstration of the requirement for cyclin D3 in a G1 phase progression.

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27 kip1 but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMPdependent mitogenesis. Here we demonstrate that transforming growth factor ␤ 1 (TGF␤ 1 ) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGFϩserum and TSH did not interfere importantly with TGF␤ receptor signaling, because they did not affect the TGF␤-induced nuclear translocation of Smad 2 and 3. TGF␤ inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGFϩserum-treated cells. TGF␤ did not inhibit c-myc expression. In TSH-stimulated cells, TGF␤ did not affect the expression of cyclin D3, cdk4, and p27 kip1 , nor the induced formation of cyclin D3-cdk4 complexes, but it prevented the TSH-induced relocalization of p27 kip1 from cdk2 to cyclin D3-cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27 kip1 away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27kip1 . It provides a new mechanism of regulation of proliferation by TGF␤, which points out the subcellular location of cyclin d-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture. INTRODUCTIONTransforming growth factor ␤ 1 (TGF␤ 1 ) is a multifunctional cytokine, member of a large family of growth and differentiation factors subdivided into three groups that include the TGF␤s, the activins, and the bone morphogenetic proteins, plus various other distantly related members such as Mü llerian-inhibiting substance. TGF␤ 1 exerts different, and often opposite, activities in controlling cell cycle progression, cell differentiation, cell adhesion, chemotaxy, and extracellular matrix deposition in a variety of cell lineages (Barnard et al., 1990;Lyons and Moses, 1990). However, in many cell types, including most epithelial cells, TGF␤ 1 is the most potent growth inhibitory polypeptide known (Roberts et al., 1985;Barnard et al., 1990). Deregulation of TGF␤ function has been implicated in carcinogenesis and the pathological processes of several human diseases. The in vitro inhibitory response is lost in many neoplastically trans...

Cell cycle proteins regulate the transitions from G1 to S and G2 to M phases. In higher eukaryotes, their function is controlled by intracellular cascades regulated by extracellular growth factors. We have studied in previously described transgenic mouse models for thyroid proliferative diseases the expression of the key proteins regulating the cell cycle by Western blotting and immunohistochemistry, and have correlated the observations with the known actions of the transgenes on the signal transduction cascades. In the adenosine A 2a receptor model, the cyclic AMP pathway, upstream of the Rb family cell division block, is constitutively activated. In the model expressing HPV 16 E7 protein, the Rb-like proteins are inhibited. Cyclin-dependent kinases cdk4, cdk2 and cdc2, and the associated cyclins D, E and A have been studied. Cyclin D3 appears as the major cyclin D subtype expressed in mouse thyroid epithelial cells in normal and transgenic mice. In the adenosine A 2a R model, all cell cycle proteins tested were accumulated. In the E7 model, all cell cycle proteins except for D-type cyclins and cdk4 were also accumulated. A similar pattern was observed in thyroids coexpressing both transgenes, suggesting a dominant e ect of E7 over the consequences of the cAMP cascade activation. The cyclin-dependent kinase inhibitors p21 cip1/waf1 and p27 kip1 were not downregulated in these proliferating thyroids which suggest other roles than the inhibition of the cell cycle progression.