Exosomes in tumor microenvironment influence cancer progression and metastasis

Exosomes are small membrane vesicles of endocytic origin with a size of 50–100 nm. They can contain microRNAs, mRNAs, DNA fragments, and proteins, which are shuttled from a donor cell to recipient cells. Many different cell types including immune cells, mesenchymal cells, and cancer cells release exosomes. There is emerging evidence that cancer-derived exosomes contribute to the recruitment and reprogramming of constituents associated with tumor environment. Here, we discuss different mechanisms associated with biogenesis, payload, and transport of exosomes. We highlight the functional relevance of exosomes in cancer, as related to tumor microenvironment, tumor immunology, angiogenesis, and metastasis. Exosomes may exert an immunosuppressive function as well as trigger an anti-tumor response by presenting tumor antigens to dendritic cells. Exosomes may serve as cancer biomarkers and aid in the treatment of cancer.     

Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCdelta in platelets

Thrombin has been known to cause tyrosine phosphorylation of protein kinase C delta (PKCdelta) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCdelta and the role of this event in platelet function. PKCdelta was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCdelta did not occur in Galpha(q)-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5'-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ', N '-tetraacetic acid), suggesting a role for Galpha(q) pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCdelta. Inhibition of tyrosine phosphorylation or the kinase activity of PKCdelta dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCdelta in a calcium- and Src-family kinase-dependent manner in platelets, with functional implications in thromboxane A2 generation.     

Thrombospondin-1 Deficiency Causes a Shift from Fibroproliferative to Inflammatory Kidney Disease and Delays Onset of Renal Failure

Thrombospondin-1 (TSP1) is a multifunctional matricellular protein known to promote progression of chronic kidney disease. To gain insight into the underlying mechanisms through which TSP1 accelerates chronic kidney disease, we compared disease progression in Col4a3 knockout (KO) mice, which develop spontaneous kidney failure, with that of Col4a3;Tsp1 double-knockout (DKO) mice. Decline of excretory renal function was significantly delayed in the absence of TSP1. Although Col4a3;Tsp1 DKO mice did progress toward end-stage renal failure, their kidneys exhibited distinct histopathological lesions, compared with creatinine level–matched Col4a3 KO mice. Although kidneys of both Col4a3 KO and Col4a3;Tsp1 DKO mice exhibited a widened tubulointerstitium, predominant lesions in Col4a3 KO kidneys were collagen deposition and fibroblast accumulation, whereas in Col4a3;Tsp1 DKO kidney inflammation was predominant, with less collagen deposition. Altered disease progression correlated with impaired activation of transforming growth factor-β1 (TGF-β1) in vivo and in vitro in the absence of TSP1. In summary, our findings suggest that TSP1 contributes to progression of chronic kidney disease by catalyzing activation of latent TGF-β1, resulting in promotion of a fibroproliferative response over an inflammatory response. Furthermore, the findings suggest that fibroproliferative and inflammatory lesions are independent entities, both of which contribute to decline of renal function.Progression of chronic kidney disease (CKD) toward end-stage renal failure (ESRF) is a prominent problem in clinical nephrology.¹ The incidence of CKD is rising, but effective therapies to halt progression of disease remain elusive.² Progression of CKD results from a complex interplay of pathologies that involve all constituents of the kidney, which makes it difficult to single out targets for effective therapeutic strategies.³The extent of so-called tubulointerstitial fibrosis is often considered to be the rate-limiting step in progression of CKD.¹ This idea is founded on histopathological analysis of large cohorts of kidney biopsies, which demonstrated that only tubulointerstitial fibrosis (which at the time was determined as the relative volume of the interstitium within a kidney biopsy section) correlates with and also predicts progression of CKD toward ESRF, irrespective of the underlying primary disease.^{4, 5, 6, 7} Widening of the tubulointerstitium, which is referred to as tubulointerstitial fibrosis, is caused by a composite of extracellular matrix (ECM) accumulation, sterile inflammation, accumulation of activated fibroblasts, and rarefaction of microvessels.¹ Although the relevance of each of these events to progression of fibrosis and CKD is hotly debated, this knowledge led to the concept that tubulointerstitial fibrosis is a common pathway of all chronic progressive kidney diseases and that effective antifibrotic therapies could potentially halt progression of CKD irrespective of the underlying disease. However, such therapies are not yet available.¹Our aim was to gain insight into mechanisms that underlie the contribution of thrombospondin-1 (TSP1) to progression of CKD. TSP1 is the most-studied member of the thrombospondin family of matricellular proteins.⁸ Previous studies have demonstrated that pharmacological suppression or genetic depletion of TSP1 attenuates disease progression in animal models of CKD.^{9, 10, 11, 12, 13} TSP1 is a 450-kDa trimeric ECM protein, which does not fulfill primarily structural roles in the matrix, but instead functions as an extracellular modulator of cell function.^{8, 14} Most prominently, TSP1 is known to inhibit angiogenesis, inhibit inflammation, activate MMP-dependent ECM turnover, and facilitate fibroblast migration and activation, all of which are considered important contributors to progression of CKD.^{8, 10} To delineate through which of its known biological activities TSP1 impacts progression of CKD, we compared progression of kidney disease of Col4a3 knockout (KO) mice (deficient in type IV collagen α3 chain) with that of Col4a3;Tsp1 double-knockout (DKO) mutant mice.¹⁵Here, we demonstrate that decrease of excretory renal function is delayed if TSP1 is absent. Furthermore, tissue analysis of plasma creatinine level–matched kidneys of Col4a3 KO and of Col4a3;Tsp1 DKO revealed that in Col4a3 KO mice disease progression is predominantly associated with fibrosis, whereas inflammation is the predominant interstitial pathology in Col4a3;Tsp1 DKO mice. We provide evidence that this altered disease progression is due to impaired activation of latent transforming growth factor-β1 (TGF-β1) in the absence of TSP1. Our findings provide evidence that both fibroproliferative injury and inflammation can independently cause expansion of the interstitium, leading to decline of excretory renal function.     

A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor.

Fetal membranes usually rupture during the process of labor. Premature fetal membrane rupture occurs not infrequently and is associated with significant fetal and maternal morbidity. The mechanisms of normal and pathologic fetal membrane rupture are not well understood. We have examined structural and biochemical changes in the rat amnion as labor approaches in order to characterize this process in normal pregnancy. Here we report that before the onset of active labor the amnion epithelial cells undergo apoptotic cell death which encompasses degradation of 28S ribosomal subunit RNA and associated P proteins and fragmentation of nuclear DNA. Concurrent with these cellular changes, the amnion type I collagen matrix is degraded with the accumulation of three-quarter length type I collagen fragments in extraembryonic fluid, characteristic of the cleavage of fibrillar collagen by interstitial collagenase. Western blot and immunohistochemical analyses confirmed that interstitial collagenase protein appears in association with the loss of amnion type I collagen. We conclude that amnion epithelial cells undergo a process of programmed cell death associated with orchestrated extracellular matrix degradation which begins before the onset of active labor. Thus, fetal membrane rupture is likely to be the result of biochemical changes as well as physical forces.     

Spatial Gradients in the Size of Inner Hair Cell Ribbons Emerge Before the Onset of Hearing in Rats

The size and locations of pre-synaptic ribbons and glutamate receptors within and around inner hair cells are correlated with auditory afferent response features such as the spontaneous discharge rate (SR), threshold, and dynamic range of sound intensity representation (the so-called SR-groups). To test if the development of these spatial gradients requires experience with sound intensity, we quantified the size and spatial distribution of synaptic ribbons from the inner hair cells of neonatal rats before and after the onset of hearing (from post-natal day (P) 3 to P33). To quantify ribbon size, we used high resolution fluorescence confocal microscopy and 3-D reconstructions of immunolabeled ribbons. The size, density, and spatial distribution of ribbons changed during development. At P3, ribbons were densely clustered near the basal/modiolar face of the hair cell where low SR-groups preferentially contact adult hair cells. By P12, the disparity in ribbon count was less striking and ribbons were equally likely to occupy both faces. At all ages before P12, ribbons were larger on the modiolar face than on the pillar face. These differences initially grew larger with age but collapsed around the onset of hearing. Between P12 and P33, the spatial gradients remained small and began to re-emerge around P33. Even by P12, we did not find spatial gradients in the size of the post-synaptic glutamate receptors as is found on afferent terminals contacting adult inner hair cells. These results suggest that spatial gradients in ribbon size develop in the absence of sensory experience.