Mini review

Thrombospondins in physiology and disease: New tricks for old dogs

Glioma stem cells (GSCs) are thought to be responsible for the initiation and progression of glioblastoma (GBM). GBM presents highly invasive growth with a very high recurrence rate, so it has become a clinical problem to be solved urgently. RNAseq demonstrates that thrombospondin 1 (THBS1) acts not only in the angiogenic core of glioma but also with a high degree of invasiveness and infiltration. Nevertheless, defects in the signaling pathway research lead to a poor prognosis in glioma patients. To investigate the relevant molecular mechanism and signal pathway of glioma stem cell behavior mediated by THBS1, U251 astroglioma cells and GSCs were taken as model cells for in vitro experiments. The biological effects of THBS1 on glioma proliferation, migration, and adhesion were evaluated using Cell Counting Kit-8(CCK8) assays, EdU incorporation assays, migration assays, Transwell assays, Western blotting, and RNAseq. We found that the knockout of the THBS1 gene by CRISPR/Cas9 promoted proliferation and migration in U251 cells and GSCs, as well as influencing cell cycle progression by regulating the TNF/MAPK/NF-κB and TGF-β/Smad signaling pathways. Moreover, U251 cells and GSCs showed different responses to THBS1 knockout, suggesting specific and potential targets for GSCs in signaling pathways mediated by THBS1.

The TGF beta-1, -2 and -3 isoforms are transcribed from different genes but bind to the same receptors and signal through the same canonical and non-canonical signal transduction pathways. There are numerous regulatory mechanisms controlling the action of each isoform that include the organ-specific cells producing latent TGF beta growth factors, multiple effectors that activate the isoforms, ECM-associated SLRPs and basement membrane components that modulate the activity and localization of the isoforms, other interactive cytokine-growth factor receptor systems, such as PDGF and CTGF, TGF beta receptor expression on target cells, including myofibroblast precursors, receptor binding competition, positive and negative signal transduction effectors, and transcription and translational regulatory mechanisms. While there has long been the view that TGF beta-1and TGF beta-2 are pro-fibrotic, while TGF beta-3 is anti-fibrotic, this review suggests that view is too simplistic, at least in adult tissues, since TGF beta-3 shares far more similarities in its modulation of fibrotic gene expression with TGF beta-1 and TGF beta-2, than it does differences, and often the differences are subtle. Rather, TGF beta-3 should be seen as a fibro-modulatory partner to the other two isoforms that modulates a nuanced and better controlled response to injury. The complex interplay between the three isoforms and numerous interactive proteins, in the context of the cellular milieu, controls regenerative non-fibrotic vs. fibrotic healing in a response to injury in a particular organ, as well as the resolution of fibrosis, when that occurs.

Impaired healing leading to the formation of ulcerated wounds is a critical concern in patients with diabetes. Abnormalities in extracellular matrix (ECM) production and remodeling contribute to tissue dysfunction and delayed healing. Specifically, diabetes-induced changes in the expression and/or activity of structural proteins, ECM-modifying enzymes, proteoglycans, and matricellular proteins have been reported. In this review, we provide a summary of the key ECM molecules and associated changes in skin and diabetic wounds. Such information should allow for new insights in the understanding of impaired wound healing and lead to the development of ECM-based therapeutic strategies.

Collagen fibrillogenesis and crosslinking have long been implicated in extracellular matrix (ECM)-dependent processes such as fibrosis and scarring. However, the extent to which matricellular proteins influence ECM protein production and fibrillar collagen crosslinking has yet to be determined. Here we show that thrombospondin 2 (TSP2), an anti-angiogenic matricellular protein, is an important modulator of ECM homeostasis. Specifically, through a fractionated quantitative proteomics approach, we show that loss of TSP2 leads to a unique ECM phenotype characterized by a significant decrease in fibrillar collagen, matricellular, and structural ECM protein production in the skin of TSP2 KO mice. Additionally, TSP2 KO skin displays decreased lysyl oxidase (LOX), which manifests as an increase in fibrillar collagen solubility and decreased levels of LOX-mediated fibrillar collagen crosslinking. We show that these changes are indirectly mediated by miR-29, a major regulator of ECM proteins and LOX, as miR-29 expression is increased in the TSP2 KO. Altogether, these findings indicate that TSP2 contributes to ECM production and assembly by regulating miR-29 and LOX.

Proteins are essential for sperm function, including their fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, vary in their functionality, which could be related to different sperm protein composition. This study aimed (i) to update the porcine sperm proteome and (ii) to identify proteins differentially expressed in mature spermatozoa from cauda epididymis and those delivered in separate ejaculate fractions. Ejaculates from nine mature and fertile boars were manually collected in three separate portions: the first 10 ml of the sperm-rich ejaculate fraction (SRF), the rest of the SRF and the post-SRF. The contents of cauda epididymides of the boars were collected post-mortem by retrograde duct perfusion, generating four different semen sources for each boar. Following centrifugation, the resulting pellets of each semen source were initially pooled and later split to generate two technical replicates per source. The resulting eight sperm samples (two per semen source) were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus scrofa taxonomy). After an ANOVA test, 32 Sus scrofa proteins showed quantitative differences (p < 0.01) among semen sources, which was particularly relevant for sperm functionality in the post-SRF. The present study showed that the proteome of boar spermatozoa is remodeled during ejaculation involving proteins clearly implicated in sperm function. The findings provide valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility.

Transforming growth factor-β (TGF-β) is a central player in fibrotic disease. Clinical trials with global inhibitors of TGF-β have been disappointing, suggesting that a more targeted approach is warranted. Conversion of the latent precursor to the biologically active form of TGF-β represents a novel approach to selectively modulating TGF-β in disease, as mechanisms employed to activate latent TGF-β are typically cell, tissue, and/or disease specific. In this review, we will discuss the role of the matricellular protein, thrombospondin 1 (TSP-1), in regulation of latent TGF-β activation and the use of an antagonist of TSP-1 mediated TGF-β activation in a number of diverse fibrotic diseases. In particular, we will discuss the TSP-1/TGF-β pathway in fibrotic complications of diabetes, liver fibrosis, and in multiple myeloma. We will also discuss emerging evidence for a role for TSP-1 in arterial remodeling, biomechanical modulation of TGF-β activity, and in immune dysfunction. As TSP-1 expression is upregulated by factors induced in fibrotic disease, targeting the TSP-1/TGF-β pathway potentially represents a more selective approach to controlling TGF-β activity in disease.