Invoking the Power of Thrombospondins: Regulation of Thrombospondins Expression

MATBIO-01044; No of Pages 14 Matrix Biology xxx (2014) xxx–xxx

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Matrix Biology

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Invoking the power of thrombospondins: Regulation of thrombospondins expression

Olga Stenina-Adognravi ⁎

Department of Molecular Cardiology, Cleveland Clinic, 9500 Euclid Ave NB50, Cleveland, OH 44195, United States article info abstract

Article history: Increasing evidence suggests critical functions of thrombospondins (TSPs) in a variety of physiological and path- Received 16 December 2013 ological processes. With the growing understanding of the importance of these matricellular proteins, the need to Received in revised form 5 February 2014 understand the mechanisms of regulation of their expression and potential approaches to modulate their levels is Accepted 8 February 2014 also increasing. The regulation of TSP expression is multi-leveled, cell- and tissue-speciﬁc, and very precise. Available online xxxx However, the knowledge of mechanisms modulating the levels of TSPs is fragmented and incomplete. This Keywords: review discusses the known mechanisms of regulation of TSP levels and the gaps in our knowledge that prevent Thrombospondin us from developing strategies to modulate the expression of these physiologically important proteins. Regulation of expression © 2014 Elsevier B.V. All rights reserved. Transcription Translation miRNA

1. Introduction important physiological and pathological processes, as well as understanding that cells do not function in isolation from their environment Thrombospondins (TSPs) are secreted extracellular proteins that be- and ECM. long to a group of matricellular proteins — proteins that are present in The human TSP protein family consists of five members (TSP-1, TSP-2, the extracellular matrix (ECM), but do not play a primary structural TSP-3, TSP-4, and TSP-5, or cartilage oligomeric matrix protein, COMP), role as other ECM proteins do. Matricellular proteins, including TSPs, which are divided into two subgroups based on their domain structure have diverse functions that integrate ECM and cells: they interact with (Adams, 2001; Adams and Lawler, 2004, 2011) (TSP-1 and TSP-2 belong a number of cell surface receptors and with a variety of ECM proteins to subgroup A, while TSP-3, TSP-4, and TSP-5 compose subgroup B). In (e.g., structural proteins such as collagens, proteases, and growth lower organisms, TSPs are represented by a single protein, as in sponge factors). The revolutionary classification of matricellular proteins pro- (Bentley and Adams, 2010), or by multiple family members, e.g., five posedbylatePaulBornstein(Sage and Bornstein, 1991; Bornstein, proteins in sea anemone (Tucker et al., 2013), that have a structure sim- 1995) has promoted a better appreciation of ECM and its role in ilar to human TSPs of subgroup B. The second subgroup A appears later in the evolutionary development, coinciding with the development of the vascular system (Bentley and Adams, 2010), and develops further as a re- flection of the development of cardiovascular and immune systems wherethisgroupplaysanimportantrole. Abbreviations: AhR, Aryl (aromatic) hydrocarbon receptor; ARE, Adenylate-uridylate- rich element; ATF-1, Activating Transcription Factor-1; CBF, CCAAT-binding factor; COMP, Important physiological functions of TSPs have been discovered in Cartilage oligomeric matrix protein; Cyp1B1, Cytochrome P450 1B1; EBOX, Enhancer box; many organs and systems [reviewed in (Henkin and Volpert, 2011; ECM, Extracellular matrix; Egr-1, Early growth response protein 1; EGRF, Early growth re- Frangogiannis, 2012; Mayer et al., 2013; Stenina-Adognravi, 2013)]. sponse factor 1; eNOS, Endothelial nitric oxide synthase; ER, Endoplasmic reticulum; H3, Regulation of angiogenesis and cancer progression (Lawler and Histone 3; HDAC, Histone Deacetylase; HGF, Hepatocyte growth factor; HuR, Human anti- Lawler, 2012), regulation of inflammation (Frolova et al., 2010; Lopez- gen R; IR, Ischemia/reperfusion; MAPK, Mitogen-activated protein kinases; MI, Myocardial infarction; MYC, Myelocytomatosis oncogene; NO, Nitric oxide; NR4A2, Nuclear receptor Dee et al., 2011; Mustonen et al., 2012; Stenina-Adognravi, 2013; subfamily 4, group A, member 2; PAH, Pulmonary arterial hypertension; PAR-1, Vanhoutte et al., 2013), modulation of immune response (Martin- Protease-activated receptor-1; SPARC, Secreted protein acidic and rich in cysteine; TGFβ, Manso et al., 2012; Miller et al., 2013), formation of myotendinous junc- Transforming growth factor beta; TRPC4, Short transient receptor potential channel 4; tions (Subramanian et al., 2007), maintenance of the myocardium TSP, Thrombospondin; UTR, Untranslated region; WT1, Wilms' tumor suppressor; YY-1, integrity and function (Schroen et al., 2004; Chatila et al., 2007; Yin Yang 1. ⁎ Tel.: +1 216 444 9057 (office); fax: +1 216 445 8204. Swinnen et al., 2009; Cingolani et al., 2011; van Almen et al., 2011a; E-mail address: [email protected]. Frolova et al., 2012; Lynch et al., 2012; Roberts et al., 2012), regulation

Please cite this article as: Stenina-Adognravi, O., Invoking the power of thrombospondins: Regulation of thrombospondins expression, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.02.001 2 O. Stenina-Adognravi / Matrix Biology xxx (2014) xxx–xxx of fibrosis (Sweetwyne and Murphy-Ullrich, 2012), and synaptogenesis (Frolova et al., 2010). Similarly, in the wall of smaller blood vessels (Risher and Eroglu, 2012) are just a few examples of their roles in TSP-3, TSP-4, and TSP-5 not only are produced by different cell types, physiology and pathology. To participate in the critical physiological but also are deposited in ECM in distinct patterns and in distinct locali- processes, TSPs have to be present in the right location at the right zation within the vessel (Frolova, in press). In tendon, both TSP-3 and time. This review summarizes the published information about the TSP-4 are abundant, but are arranged in fibers of different orientations, regulation of the expression of TSPs. stressing the distinctions in their functions (Frolova, in press). When TSPs are expressed in the same structures at the same time [e.g., TSP-4 2. Similarities and distinctions between TSP family members and TSP-5 in tendon (Frolova, in press)], it is still unclear why both are required and what differential properties of tissue they support. All TSPs share a high degree of homology at the protein level, espe- Differences in the expression profiles of individual TSPs in cancers cially in their “signature domain”—the C-terminal half of the human and other cells and tissues [e.g., (Carron et al., 2000; Agah et al., 2002; protein that includes 3–4EGF-likerepeats[1–5 in other species Bornstein et al., 2004)] suggest that TSPs have differential functions in (Adams and Lawler, 2011)], the Ca2+-binding domains with up to 26 pathological processes, despite shared homologous domains and li- metal binding sites per protein subunit, and the globular C-terminal do- gands. Several gene expression studies demonstrated opposite profiles main [reviewed in (Adams and Lawler, 2011)]. Table 1 summarizes the for TSP-1 and TSP-4 in brain (Bredel et al., 2005; Liang et al., 2005; extent of homology between five TSPs. However, apart from the protein Sun et al., 2006) and breast (Sorlie et al., 2001; Turashvili et al., 2007; coding regions of the TSP genes (THBS), there is no homology in their Ma et al., 2009) cancers. Examination of the datasets from these studies DNA sequence, and the regulatory parts of TSP genes, either the reveals that TSP-4 upregulation is accompanied by downregulation of untranslated regions of mRNA (UTR) or the gene promoters, are dis- TSP-1 in the tumor samples (www.oncomine.org), clearly indicating tinctly different at the first glance (Adolph et al., 1997). Search for com- distinct, probably even opposite, functions for these two proteins. Unex- mon DNA sequence motifs using SCOPE motif finder (http://genie. pectedly, TSP-2, belonging to subgroup A and sharing high homology dartmouth.edu/scope)(Carlson et al., 2007; Chakravarty et al., 2007) with TSP-1, exhibits a profile similar to the TSP-4 profile — it is upregu- and comparison of the positions of common DNA sequence motifs in lated in both brain and breast cancers, with the exception of one data set the promoters of five TSPs reveal some similarities between TSP pro- (Bredel et al., 2005). In some datasets TSP-2 was in top 1% of upregulat- moters and suggest that in some conditions two or more TSPs may be ed genes, together with TSP-4. These comparisons of the three TSP pro- regulated by the same stimuli simultaneously (Fig. 1). However, there files from the same datasets clearly demonstrate that the three TSPs are no experimental data to support the predicted similarity of the play important distinct roles in cancer growth, and the regulation of DNA motifs. their expression is an efficient mechanism to support these still poorly The five proteins are located on different chromosomes (human understood functions (with the exception of TSP-1, whose downregula- THBS1 on chromosome 15 and mouse Thbs1 on chromosome 2, tion is known to support angiogenesis in tumors). TSP-5 was upregulat- human THBS2 on chromosome 6 and mouse Thbs2 on chromosome ed similar to TSP-4 in multiple datasets from breast cancer studies 17, human THBS3 on chromosome 1 and mouse Thbs3 on chromosome (www.oncomine.org), while TSP-3 data were inconsistent, and both 3, human THBS4 on chromosome 5 and mouse Thbs4 on chromosome 13, down-regulation and up-regulation were observed (Ramaswamy human THBS5 on chromosome 19 and mouse Thbs5 on chromosome 8). et al., 2003; Yu et al., 2008). In most tissues, TSPs are expressed at a low level compared to other The expression studies in human samples and in animal tissues non-structural ECM proteins, e.g., SPARC, tenascin C and fibronectin clearly suggest distinct functions and roles in physiological and patho- (Fig. 2)(Su et al., 2002). Similar to TSPs, tenascin C and SPARC are logical processes for five TSPs. However, the molecular mechanisms representatives of the matricellular protein family. SPARC and fibronec- responsible for the differences in TSPs functions still have to be ad- tin regulate collagen deposition and ECM assembly and support the in- dressed and confirmed by examining specific functions of all five TSPs teraction between cells and ECM, similar to TSPs. These proteins have and by studying their interactions with cells and ligands. Precise regula- similar functions in disease regulation: e.g., they support interaction of tion of TSP expression is especially important in a view of TSP homology cancer cells with stromal cells. and a number of shared ligands. Emphasizing the potent effect of TSP presence in the tissue and the need to tightly regulate their expression, there are rapid mechanisms 3. Mechanisms of regulation of TSP expression upregulating TSPs at the transcriptional level (Raugi et al., 1987; Stenina et al., 2003a; Dabir et al., 2008) and mechanisms rapidly Most of the information about the regulation of TSP expression degrading mRNA or blocking its translation into a protein (Janz et al., comes from descriptive studies that either used array type experiments 2000; Dews et al., 2006; Bhattacharyya et al., 2008; Sanghamitra to identify highly upregulated or downregulated genes in tissues of Bhattacharyya et al., 2012). The proteins appear to be unstable after interest or specifically looked at the TSP expression (in most cases they are secreted, stressing the importance of timely elimination of expression of TSP-1), based on known functions of TSPs. However, a TSPs from the ECM and cell environment (Murphy-Ullrich and Mosher, few reports address specific molecular mechanisms of the regulation 1987a, 1987b; Manni et al., 2007). In adult organisms, upregulation of of TSP expression in cells and tissues. TSPs is associated with specific stages of wound healing [e.g., (Raugi et al., 1987; Reed et al., 1993; DiPietro et al., 1996; Kyriakides et al., 3.1. Transcriptional regulation 1999; Streit et al., 2000; Agah et al., 2002)] and tissue remodeling [e.g., (Stenina et al., 2003b; Rysa et al., 2005; Mustonen et al., 2008; 3.1.1. TSP-1 Frolova et al., 2010; Cingolani et al., 2011; Lynch et al., 2012; Pohjolainen et al., 2012)], in which it can be either protective and 3.1.1.1. Positive regulation. The first analysis of TSP-1 promoter and the beneficial or detrimental. identification of the serum response element were reported by Distinct localization of TSPs in tissues suggests differential functions, Framson and Bornstein (1993).Theidentified serum response element despite the high homology between proteins and a number of shared is bi-partite and includes a distal element at the position −1280 and a cell surface receptors and binding partners. Even when two or more proximal element with NF-Y binding site at the position −65 (Fig. 3). TSPs are found in the same tissue, they are localized to the different Interestingly, the proximal element is different in the mouse TSP-1 pro- cell types or structures within the tissue. For example, both TSP-3 moter: it harbors Egr-1 binding site in the position of human NF-Y site and TSP-5 are present in the atherosclerotic lesion of ApoE−/− mice, and does not participate in serum response. The Egr-1 binding site but they are clearly produced by different cell types in the lesion maintained constitutive activity of the promoter acting in concert with

Table 1 For example, in response to high glucose, TSP-1 mRNA is upregulated Homology of human TSP proteins [calculated using algorithm described in (Huang and in major cell types of the vascular wall — endothelial cells, smooth Miller, 1991)]. muscle cells and fibroblasts (Stenina et al., 2003a). However, the time TSP-1 TSP-2 TSP-3 TSP-4 of upregulation is distinctly different — in endothelial cells upregulation TSP-1 can be detected earlier than in smooth muscle cells. The analysis of the TSP-2 62.6 promoter revealed that in two cell types the promoter activity is regu- TSP-3 52.2 52.9 lated by different transcriptional protein complexes. In endothelial TSP-453.952.661 cells, a proximal regulatory region of the promoter binding aryl hydro- TSP-5 53.6 54.4 65.3 69.9 carbon receptor is sufficient to drive the transcription (Dabir et al., 2008; Raman et al., 2011), while in smooth muscle cells the same promoter region is interacting with a distal part of the promoter the adjacent GC-rich region binding SP1 (Shingu and Bornstein, 1994). through the formation of a single protein complex between transcrip- The importance of the first intron for the efficient transcription of the tion factors binding to the proximal and to the distal regions (Raman gene was noted (Laherty et al., 1989): the deletion of this region results et al., 2007; Raman et al., 2011). The regulation is determined by a in 4-fold decrease in the reporter production, suggesting a cis-acting cell-type-specificprofile of the expression and the activation of positive element in the first intron. transcription factors. AP-1 binding to TSP-1 gene promoter mediates the activation of Transcriptional upregulation of TSP-1 in response to high glucose is the promoter in response to protein kinase C (PKC) activation in probably the most dissected pathway among all the pathways regulat- human hepatoma cells (Kim et al., 2001). However, a similar study ing TSP-1 expression. TSP-1 has been implicated in the development of the effects of PKC activation in porcine aortic endothelial cells re- of vascular complications of diabetes, and its mRNA was dramatically sulted in opposite conclusions: a specific region of the promoter was upregulated in hyperglycemia in various tissues and organs (Poczatek responsible for the downregulation of the promoter activity (Kim et al., 2000; Stenina et al., 2003a; Wang et al., 2003; Daniel et al., and Hong, 2000). The cell-specificity is a very common theme in 2004; Wang et al., 2004; Wang et al., 2006; Zhou et al., 2006; the studies of TSP expression. The cell-and tissue-specific differences Belmadani et al., 2007; Bhattacharyya et al., 2008; Raman et al., in regulation reflect the multi-functional nature of these proteins 2011; Chavez et al., 2012; Sanghamitra Bhattacharyya et al., 2012; and the importance of a very strictly localized and timely production Sweetwyne and Murphy-Ullrich, 2012). The initial observation of and elimination of TSPs. upregulation of TSP-1 in response to high glucose in mesangial cells The cell-specificity of TSP-1 gene promoter regulation was reported (Poczatek et al., 2000) was later confirmed in multiple cell types and tis- in several studies. The activity of a promoter is determined not only by sues. The precise transcriptional mechanisms appear to vary depending its sequence, but also by the availability of target binding sites in a spe- on the cell type, with several transcriptional factors and regulatory re- cific cell type, presence of transcription factors capable of binding the gions being constant independently of the cell type — e.g., upstream promoter, and, finally, cell-specific signals to activate the transcription stimulatory factors (USFs) (Wang et al., 2004) that are common media- factors or protein co-activators. The regulatory complex assembled on tors of glucose effects. Another transcription factor activated by high TSP-1 gene promoter is often cell-specific, and the promoter regions glucose and essential for TSP-1 gene promoter activation is aryl hydro- involved in regulation are also different depending on the cell type. carbon receptor, or AhR (Dabir et al., 2008; Raman et al., 2011). The

Fig. 1. Common DNA sequence motifs in the promoters of TSPs. The analysis of the promoters of TSP-1, TSP-2, TSP-3, TSP-4 and COMP was performed using SCOPE motif finder (http://genie.dartmouth.edu/scope)(Carlson et al., 2007; Chakravarty et al., 2007). Motifs positioned similarly in the promoters of two or more TSPs are shown in the figure (6 out of 20 most significant matches).

Fig. 2. Expression of TSPs in human tissues and organs, comparison to tenascin C, SPARC and fibronectin expression. The figure was prepared using the data from the database http://expression.gnf.org (Su et al., 2002). Organs from left to right: cerebellum, whole brain, cortex, caudate nucleus, amygdala, thalamus, corpus collosum, spinal cord, whole blood, testis, pancreas, placenta, pituitary gland, thyroid, prostate, ovary, uterus, DRG, salivary gland, trachea, lung, thymus, spleen, adrenal gland, kidney, liver, and heart. activation of the AhR pathway may provide a mechanistic connection factor that mediates many effects of hypoxia in complexes with Arnt). between glucose and hypoxia regulatory pathways (AhR is a ligand for AhR is a member of the Clock family of proteins and can form complexes HIF1β or Arnt, whose other well studied ligand is HIF1α, a transcription with several other proteins from this family that regulate circadian rhythms. The activation of AhR by glucose may provide insights into the well-known connection between food intake and maintenance of the circadian rhythms. TSP-1, as a major and highly upregulated target of AhR, may play a role in this regulation. Increasingly appreciated significance of TSP-1 function in metabolic disorders (Moura et al., 2008; Li et al., 2011) prompted an investigation of the transcriptional regulation of TSP-1 gene by leptin, a hormone implicated in the development of obesity and diabetes. Leptin induced TSP-1 at the transcriptional level through the JAK2/ERK/JNK-dependent mechanism (Chavez et al., 2012). The promoter of TSP-1 gene has an active Egr-1 binding site. The promoter activity is upregulated by Egr-1-inducing stimuli (Moon et al., 2005). TSP-1 mRNA can be very rapidly (within 10–15 min) upregulated in response to injury, both in vivo and in vitro (Stenina Fig. 3. Promoter of TSP-1: regulatory regions and experimentally confirmed binding sites et al., 2003a), and, although not investigated, this upregulation most for transcription factors. probably requires Egr-1, one of the first-response transcription factors.

Egr-1 is responsible for transcriptional upregulation of TSP-1 by transcriptional factor Id1 that is known to regulate tumor angiogenesis thrombin, together with MYC, in a thrombin receptor PAR-1, G(i/o), (Volpert et al., 2002). G(q), EBOX/EGRF-signaling cascade (McLaughlin et al., 2005). Importantly, transcriptional repression is a common mechanism TSP-1 is a potent anti-angiogenic protein, and most of the reports on regulating the expression of TSP-1 — it results in a rapid effect, because its expression and transcriptional regulation are related to the regula- both the protein and mRNA of TSP-1 are unstable (Raman et al., 2007; El tion of angiogenesis in various tissues and pathological conditions. It is Btaouri et al., 2011; McGray et al., 2011a; Raman et al., 2011; Chavez difficult to judge whether the focus on its anti-angiogenic function is et al., 2012). due to the fact that this is truly the main function of TSP-1, whether MicroRNA regulation is usually associated with the modulation of this is due to the current popularity of the angiogenesis topics, or mRNA translation or stability. However, miR-182 was reported to whether this is a result of a lack of sufficient understanding and knowl- regulate the transcription of TSP-1 gene in colon cancer by modulating edge about other functions of TSP-1. TSP-1 inhibits angiogenesis even in the function of transcription factors Egr-1 and Sp-1 function and their the presence of pro-angiogenic factors (Almog et al., 2006; Zaslavsky binding to THBS1 promoter (Amodeo et al., 2013). et al., 2010; Roudier et al., 2013). Both TSP-1 and TSP-2 were downregulated in Akt−/− mice, and this CCAAT box binding CCAAT-binding factor (CBF) mediates the effect downregulation was responsible for the increased angiogenesis in these of histone deacetylase (HDAC) inhibitors in the regulation of angiogen- mice. The TSP-2 promoter inactivation by dominant negative Akt sug- esis and tumor growth: acetylated CBF specifically binds to TSP-1 gested that the regulation is transcriptional, but the exact mechanism promoter, and its activity is increased by the binding of acetylated H3 has not been addressed (Chen et al., 2005). Although the studies identi- (Kang et al., 2008). fying the signals that lead to the transcriptional upregulation are infor- Recently, the role of TSP-1 in inflammation has been actively studied mative in defining the stimuli regulating the expression of TSPs, without and confirmed in several models (McMorrow et al., 2013). Transcrip- addressing the molecular mechanism and delineating the promoter tional regulation of TSP-1 in chronic inflammation was addressed in in- elements involved in the regulation, it is impossible to know whether flammatory joint disease (McMorrow et al., 2013): the orphan receptor the observed effects are direct or mediated by an autocrine mediator 4A2 (NR4A2) was responsible for the downregulation of the promoter and whether the mechanism of the regulation in these examples is activity, and the anti-tumor necrosis factor treatment, which decreases truly transcriptional. NR4A2, increased TSP-1 expression and suggested that TSP-1 plays a role in resolution of inflammation. 3.1.2. TSP-2 Many studies reported the stimuli regulating transcription without The promoters of human and mouse TSP-2 genes were analyzed addressing specific transcriptional mechanisms and transcription and compared in (Adolph et al., 1997). Interestingly, tissue-specific factors involved. differences in transcription sites were found, resulting in transcripts For example, TGFβ1 and TGFβ2stimulatedTSP-1andTSP-2produc- of different lengths depending on the tissue. This was the only report tion in bovine adrenocortical cells, and the effect was blocked by the considering the features of THBS2 promoter. Putative transcription transcription inhibitors (Negoescu et al., 1995). factor binding sites were analyzed, and comparison to THBS1 promoter did not reveal any similarities between two genes in the promoter regions. 3.1.1.2. Negative regulation. TSP-1 gene promoter appears to have an in- hibitory element between −300 and −750 bp positions, as was report- 3.1.3. Other TSPs ed in two independent studies (Kang et al., 2004; Dabir et al., 2008). Dr. Bornstein's group reported on the promoter of THBS3 as well. Shorter promoter deletion constructs are significantly more active, as Interaction between THBS3 gene and adjacent metaxin gene was are the longer constructs, both constitutively and in response to stimuli. revealed (Collins et al., 1998), and the active SP1-binding elements The promoter was found to be sensitive to treatment with nickel were identified in the common regulatory region of the two genes (Salnikow et al., 1994): nickel downregulated the activity of the pro- (Collins and Bornstein, 1996). Analysis of the transcription initiation moter and decreased the expression of endogenous TSP-1 in cultured sites revealed alternative transcripts (Adolph and Bornstein, 1999). hamster embryo cells. Activating transcription factor-1 (ATF-1) was Since the last publication in 1999, there have been no additional reports identified as a component of the protein complex that bound to a on the regulation of THBS3 transcription. negative regulatory site in the mouse TSP-1 gene promoter (Salnikow There is very little information about the transcriptional regulation et al., 1997). ATF-1 binding to the region between positions −1210 of TSP-4 and TSP-5 genes, despite their reported importance in several and −1123 of the promoter harboring a cAMP-responsive element physiological and pathological processes (Hecht et al., 1995; Mustonen (CRE) induced the repression of the promoter in response to hepatocyte et al., 2008; Eroglu et al., 2009; Frolova et al., 2010; Cingolani et al., growth factor (HGF) and played a key role in tumor progression 2011; Frolova et al., 2012; Lynch et al., 2012; Risher and Eroglu, 2012). triggered by HGF (Ghoneim et al., 2007). The promoter of TSP-5 (COMP) has been analyzed, and a region required Further analyses of THBS1 promoter identified many active binding for the activity in chondrocytes was identified within 375 bp of the sites for transcription factors that tightly regulate TSP-1 expression at translational start site, as well as enhancer elements between −1.0 kb the transcriptional level. YY-1 binds to a site at the position −440 of and −1.7 kb (Posey et al., 2005). the promoter, which results in an interaction that can be weakened by the increased c-Jun levels, suggesting that the interaction of YY- and 3.2. Alternative splicing c-Jun decreases the promoter activity (Kang et al., 2004). The well-documented suppression of TSP-1 production in tumors With a large number of exons in TSP genes (N20 in most TSPs), the (upregulating angiogenesis to promote tumor growth) includes tran- possibility of alternative splicing and regulation of expression and func- scriptional downregulation: e.g., the product of Wilms' tumor suppres- tions by this mechanism is plausible. However, alternative splicing and sor gene, WT1, binds to the −210 region of the TSP-1 gene promoter its potential significance in regulation of TSPs have not been seriously and represses THBS1 transcription (Dejong et al., 1999), while tumor addressed to date. Several publications reported alternative forms of suppressor protein p53 activates TSP-1 promoter (Su et al., 2010). TSP-2 and TSP-3 (Adolph, 1999; Adolph and Bornstein, 1999). A produc- Another mechanism used by cancers to prevent TSP-1 promoter action of a Lumbar-disk-herniation-associated form of TSP-2 is due to a tivation is hypermethylation of the promoter that has been detected in polymorphism and the alternative splicing of TSP-2 that results in a variety of cancers [e.g., (Kanai et al., 2001; Yang et al., 2003; Liu et al., skipping exon 11 (Hirose et al., 2008). Lack of exon 11 causes decreased 2005; Lindner et al., 2013)]. TSP-1 promoter is also a target for a interaction of TSP-2 with metalloproteinases. Alternative splicing of

TSP-4 gene associated with change in cell motility was reported in production of a protein may not always reflect the increased levels cancer cells (Lee et al., 2008). of mRNA [e.g., (Manni et al., 2007; Bhattacharyya et al., 2008; Multiple TSP forms of different sizes are routinely observed Sanghamitra Bhattacharyya et al., 2012)]. As more examples of such (e.g.,Stenina et al., 2003a, 2005), but the origin of these forms has discordant levels of protein and mRNA accumulate, we will be forced not been thoroughly explored, and the possibility of the alternative to address the translational regulation of TSPs. splicing has not been excluded. TSP-1 mRNA is associated with rich in adenines and uracils ARE binding protein HuR. HuR facilitates translation, and it has been co- 3.3. Regulation of mRNA stability precipitated with TSP-1 mRNA from the cultured MCF7 breast cancer cells (Caux et al., 1991). Another example of translational regulation of As has been mentioned above, both TSP-1 mRNA and protein have a TSP-1 production is translational silencing of TSP-1 mRNA in response short half-life. The short half-life of TSP-1 mRNA is due, at least in part, to high glucose (Bhattacharyya et al., 2008; Sanghamitra Bhattacharyya to AU-rich elements in the 3′ untranslated regions (3′UTR) of the TSP-1 et al., 2012) that is tissue-specific and regulated by a cell-specific mRNA (McGray et al., 2011a, 2011b). Unusually large regulatory 3′UTR induction of miR-467 and its binding to TSP-1 3′UTR. of TSP-1 and TSP-2 mRNAs suggests extensive post-transcriptional regulation at the level of mRNA (Table 2). The rest of TSPs have an average 3.5. Post-translational regulation — secretion sized UTR; however, this does not exclude regulation of mRNA stability or translation. UTR of all TSPs has putative target binding sites for TSPs are secreted extracellular matrix proteins. Although some of miRNAs. There is no information on the regulation of TSP-3, TSP-4, them, e.g., TSP-1, TSP-4 and TSP-5, perform important functions inside and TSP-5 expression at the level of mRNA, but several reports de- the secretory pathways (Hecht et al., 1998, 2001; Hecht and Sage, scribed the regulation of TSP-1 and TSP-2 through the modulation of 2006; Lynch et al., 2012), most of the widely known functions rely on mRNA stability or translation. the presence of TSPs outside the cell. TSP-1 mRNA was destabilized by the activation of myc oncoprotein The regulation of secretion of pre-made protein is a very fast and a (Janz et al., 2000). Further analysis of this mechanism revealed that de- very efficient way to regulate the protein availability, and this type of stabilization is a result of increased expression of miR-17-92 family that regulation is used by the cells to control the extracellular TSP levels. binds TSP-1 mRNA (Dews et al., 2010). The decrease in the levels of the TSP-1 can be retained inside the cells, and the secretion depends on members of this family of miRNA, miR-18/19, was associated with the Ca2+ regulation: TSP-1 retention inside the cells can be induced increased TSP-1 level in cardiomyocytes of aging hearts, identifying by a calcium chelator and is regulated in renal cell carcinoma cells by the aging process (van Almen et al., 2011b). p53-responsive miR-194 TRPC4 calcium channel expression (Veliceasa et al., 2007). TSP-1 is a decreased the levels of mature TSP-1 mRNA despite the increased levels major component of platelet alpha granules: pre-synthesized protein of the primary transcript, prevented the production of TSP-1 protein, is released upon platelets activation (Lawler and Slayter, 1981; Dawes and promoted angiogenesis in colon cancers (Sundaram et al., 2011), et al., 1983). Macrophages also regulate TSP-1 secretion: the secretion presumably, due to destabilization of TSP-1 mRNA. is induced by LPS, and the threshold for the level of secretion is regulated Increased TSP-1 mRNA stability was detected in response to by the environmental signals, including the mediators of the adaptive heat shock (Kang et al., 2006), and a region of TSP-1 3′UTR respon- immune system, Th1 and Th2 cytokines (Fordham et al., 2012). TSP-1 sible for the increased stability was identified as 968–1258 from secretion depends on cell density in culture: lower density results in the stop codon. TGFβ also increased TSP-1 expression in cultured higher secretion of TSP-1 by fibroblasts, endothelial and smooth muscle osteosarcoma cells by stabilizing mRNA via p38 MAPK signaling cells without change in protein synthesis (Mumby et al., 1984). (Okamoto et al., 2002). Both TSP-4 and TSP-5 appear to have additional intracellular There are no mechanistic reports describing the effects on TSP-2 functions in the secretory pathways: TSP-5 can associate with its mRNA stability. Although there are examples of post-transcriptional co-secreted extracellular ligands and ER chaperones (Hecht et al., regulation of TSP-2 [e.g., in (Bein et al., 1998) describing the effect of 1998, 2001; Hecht and Sage, 2006), and TSP-4 bind a transcriptional c-myb on TSP-2 expression via a post-transcriptional mechanism], the factor Aft6α promoting its translocation to the nucleus (Lynch et al., details of this regulation remain unknown. 2012). The latter function is shared with TSP-1 and is thought to be a common feature of TSPs in protecting and augmenting ER function 3.4. Regulation of mRNA translation (Lynch et al., 2012).

Due to the complexity of translational mechanisms and lack of 4. Descriptive studies of TSP expression detailed knowledge of these mechanisms, as well as to the requirement of speciﬁc skills and experience for the experimental manipulations More than 2400 publications address the expression of TSPs in dif- with RNA, investigation of translational regulation of TSPs has not ferent tissues and various conditions. The ﬁndings clearly indicate the been the most popular of research topics. The large size of TSP mRNA importance of the altered expression in physiology and pathology and and, especially, the long regulatory UTR of TSP-1 and TSP-2 may have stress the need to develop understanding of the mechanisms that con- discouraged this type of study. However, studies of translational regula- trol the expression. Most of the information about the expression of tion of TSPs may prove very rewarding: there are multiple independent TSPs comes from descriptive studies, in which the mechanisms of regu- observations that increased levels of mRNA do not guarantee increased lation have not been addressed. This information is valuable in identify- production of the corresponding protein and that the increased ing physiological and pathological processes dependent on TSPs and in providing basis for future studies of precise mechanisms. Among all the descriptive studies, the most valuable are the ones where protein Table 2 levels have been assessed. Whenever only the mRNA levels were evalu- Size of untranslated regions of mRNA of TSPs. ated, there is always a possibility that they do not translate into protein 5′UTR 3′UTR production or that the produced protein is not secreted, as seems to be a

TSP-1 179 2130 frequent occurrence with TSPs. TSP-2 320 2061 Although an overview of conditions resulting in altered expression TSP-3 21 234 of TSPs is not the goal of this article, several major themes in the regula- TSP-4 191 156 tion of TSPs are very prominent and must be mentioned: growth and TSP-5 36 161 remodeling of tissues, altered angiogenesis and cancer, ischemia and

Please cite this article as: Stenina-Adognravi, O., Invoking the power of thrombospondins: Regulation of thrombospondins expression, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.02.001 O. Stenina-Adognravi / Matrix Biology xxx (2014) xxx–xxx 7 reperfusion, activation of TGFβ,inflammation and immune response, (Scott-Burden et al., 1990). TSP-2 expression is downregulated by aging, and embryonic development (Table 3). Any of these pathological Cyp1B1, and this regulation is important for a proper capillary mor- or physiological situations consistently result in or are caused by the phogenesis in a model of retinopathy of prematurity (a neovascular change in the levels of one or several TSPs. As usual, TSP-1 expression response during oxygen-induced ischemic retinopathy)(Tang et al., has been better studied than the expression of the other four TSPs, 2009). The activity of endothelial nitric oxide synthase (eNOS) neg- although many recent reports repeatedly find the associations of atively correlated with TSP-2 levels: deficiency in eNOS resulted in in- TSP-4 expression and pathological changes. creased levels of TSP-2 and decreased angiogenesis in a mouse model, Altered expression of TSPs during the growth and remodeling of tis- and NO repressed the promoter of TSP-2 (MacLauchlan et al., 2011). sues is associated either with the need for angiogenesis, matrix remod- However, the precise transcriptional mechanisms were not dissected eling, or the direct stimulation of cell growth. TSP-4 is upregulated in in these studies. the mouse heart in response to pressure overload and in human The extent of angiogenesis after ischemia and reperfusion (IR) corre- hypertrophied hearts. It has a protective function regulating cardiomyo- lates with the successful repair process in tissues and with the patient cyte function and production of extracellular matrix (Gabrielsen et al., survival. Elevation of TSP-1 levels was detected after IR of the brain, 2007; Mustonen et al., 2008; Mustonen et al., 2010; Cingolani et al., myocardium, lung, and kidney (Lin et al., 2003; Frangogiannis et al., 2011; Melenovsky et al., 2011; Frolova et al., 2012; Lynch et al., 2012). 2005; Sezaki et al., 2005; Lario et al., 2007; Sage et al., 2008). While TSP-4 is expressed in atherosclerotic lesions and promotes atheroscle- the expression of TSP-2 was elevated at the peak of angiogenesis rotic process by attracting and retaining macrophages in the lesion (2 weeks after reperfusion), the expression of TSP-1 was dramatically (Frolova et al., 2010). Thbs4 is responsive to the ischemic injury in the elevated early after the event and did not seem to represent a response brain cortex and plays an important role in post-injury astrogenesis to the angiogenic process. Although it is unclear what the role of TSP-1 is (Benner et al., 2013). THBS4 expression increases with age in the brain in tissues just hours after IR, it appears to have a protective effect in the gray matter and may be associated with Alzheimer's disease and altered heart: e.g., myocardial infarction (MI) patients with higher levels of TSP- inflammatory response (Cagliani et al., 2013). Increased TSP-1 expres- 1 in the platelet-poor plasma had a better outcome and fewer adverse sion is also often found in tissue growth and remodeling, e.g., in embryo cardiac events within 14 months after MI (Kaiser et al., 2013). In animal fibroblasts in response to c-Jun stimulation (Mettouchi et al., 1994). In- models of MI, TSP-1 mRNA was induced only 1 h of ischemia and was creased levels of TSP-1 and TSP-2 have been reported in remodeling elevated 3–7 days after reperfusion. TSP-1 was localized to the infarc- large arteries in atherosclerotic lesions and after injury (Stenina et al., tion border zone and reduced inflammation and granulation tissue 2003a; Moura et al., 2008; Frolova et al., 2010; Pohjolainen et al., 2012). formation protecting non-infarcted myocardium from fibrosis (Sezaki Inflammation and immunity is an emerging field in TSP studies et al., 2005; Kaiser et al., 2013). Interestingly, in kidneys increased (Yabkowitz et al., 1993; Riessen et al., 1998; Vallejo et al., 2000; Zhao expression of TSP-1 early after IR (only 3–12 h, returning to the base et al., 2001; Lange-Asschenfeldt et al., 2002; Manna and Frazier, 2003; line by 48 h) mediated the injury, and Thbs1−/− mice showed signifi- Frangogiannis et al., 2005; Pluskota et al., 2005; Grimbert et al., 2006; cant protection from the damage after IR injury and from the renal fail- Lopez-Dee et al., 2011; Papageorgiou et al., 2012). Although functions ure (Thakar et al., 2005). Clearly, the function and the effect of the of TSPs in immune response and inflammation are not completely un- altered expression of TSPs depend on the injury mechanism and the derstood, these proteins are clearly important for the adequate immune specific cellular event characteristic for a tissue. response and the resolution of the inflammation. TSP-1 is a major activator of TGFβ1, and there is a reciprocal feed- Multiple studies documented changed expression of TSP-1 and TSP- back mechanism maintaining and amplifying the circuit: TGFβ was 2 associated with altered angiogenesis and cancer growth. Decreased identified as TSP-1 expression regulator increasing the production of TSP-1 and/or TSP-2 expression correlates with increased vascularity TSP-1 in multiple studies (Bein et al., 2004; Flugel-Koch et al., 2004). in non-small cell lung cancers (Oshika et al., 1998), colon cancer As a major activator of TGFβ1(Crawford et al., 1998), TSP-1 is often (Tokunaga et al., 1999; Kawakami et al., 2001), invasive cervical cancer expressed in tissues producing TGFβ1, e.g., in cancers with high TGFβ1 (Kodama et al., 2001; Wu et al., 2004), glioma cells (Harada et al., 2003), levels (Kawataki et al., 2000) and in embryonic development (Melnick in cytomegalovirus (CMV) infection (Cinatl et al., 1999), and many et al., 2000). TSP-1 gene mutation was identified in a family with the other situations where angiogenesis is increased. familial pulmonary arterial hypertension (PAH) (Maloney et al., 2012). Interestingly, the expression of group B thrombospodins (TSP-3, The ability of the mutant Asp362Asn TSP-1 to activate TGFβ1was TSP-4 and TSP-5) increases in cancers. Increased levels of TSP-3 were as- reduced to 1/2 of the activity of the wild-type TSP-1. TSP-1 contributes sociated with poor prognosis in osteosarcoma patients (Dalla-Torre to the development of PAH in more than one way: the levels of et al., 2006). TSP-4 expression was increased in invasive breast cancer TSP-1 are increased in PAH patients and in animal models of PAH, and was suggested to facilitate the invasion of tumor cells (Amy et al., and the interaction of TSP-1 with its receptor CD47 disrupts the 2013). It was differentially expressed in lobular versus ductal breast constitutive interaction between CD47 and Caveolin-1, causing tumors (Korkola et al., 2003). THBS4 was found to be a powerful marker increased eNOS-dependent superoxide production and oxidative of diffuse-type gastric adenocarcinomas: its expression in fibroblasts stress (Bauer et al., 2012). was stimulated by tumor cells (Forster et al., 2011). Based on the Levels of TSPs increase in multiple tissues with age (Swinnen et al., existing data, THBS4 expression is a selective marker for specificcancers, 2009; Frazier et al., 2011; van Almen et al., 2011b; Cai et al., 2012; although the regulation and the significance of the increased levels have Rogers et al., 2012; Starr et al., 2013). The significance of this increase not been explored. in TSP production is still unclear, although in some tissues it appears Many stimuli able to reduce TSP-1 levels have been identified: to be beneficial (Swinnen et al., 2009; Frazier et al., 2011; Frolova e.g., all-trans-retinoid acid in smooth muscle cells (Axel et al., 2001); et al., 2012; Rogers et al., 2012). In others, TSPs are associated with hepatocyte growth factor/scatter factor that mediates angiogenesis aging-related pathological changes (Agah et al., 2004; van Almen through positive VEGF and negative TSP-1 regulation (Zhang et al., et al., 2011b; Cai et al., 2012; Roberts et al., 2012). 2003); shear stress (Bongrazio et al., 2006), and hypoxia (Bienes- While the significance of the altered expression of TSP-1 and TSP-2 Martinez et al., 2012). Increase in TSP-1 levels is often detected in asso- is well understood in most situations, and specific consequences are ciation with decreased angiogenesis and ischemia, e.g., in chronic leg expected (e.g., altered angiogenesis, matrix remodeling, TGFβ activa- ischemia (Favier et al., 2005). Increased levels of TSP-1 mRNA were tion), we don't have a clear understanding of the significance of altered detected upon stimulation with dexamethasone (Flugel-Koch et al., expression of group B TSPs in the majority of cases. Studies of the signals 2004); progesterone (Mirkin and Archer, 2004); high glucose (Poczatek and molecular pathways regulating their expression would greatly in- et al., 2000; Stenina et al., 2003a), thrombin and angiotensin II crease our understanding of TSP associations with specific pathological

Table 3 Summary of examples of pathological and physiological conditions associated with altered expression of TSPs (references to published reports). Green — upregulated expression, red — downregulated expression, blue — both the upregulated expression and downregulated expression have been reported.

TSP-1 TSP-2 TSP-3 TSP-4 TSP-5 (COMP)

Cingolani et al., (2011); Frolova et al., (2012); Schroen et al., (2004); Gabrielsen et al., (2007); Swinnen et al., (2009); Lynch et al., (2012); Heart hypertrophy Wang et al., (2003a) van Almen et al., (2011a) Melenovsky et al., (2011); Mustonen et al., (2008); Mustonen et al., (2010); Rysa et al., (2005)

Frolova et al., (2010); Moura et al., (2008); Frolova et al., (2012); Atherosclerosis Frolova et al., (2010) Frolova et al., (2010) Frolova et al., (2010) Reed et al., (1995); Pohjolainen et al., (2012) Riessen et al., (1998)

Sweetwyne and Murphy- Pohjolainen et al., (2012); Fibrosis Ullrich, (2012) Reinecke et al., (2013)

Chen et al., (1999); Miano et al., (1993); Raugi et al., (1990); Arterial injury Roth et al., (1998); Sajid et al., (2001); Stenina et al., (2003)

Favier et al., (2005); Frangogiannis et al., (2005); Kaiser et al., (2013); Ischemia and reperfusion Lario et al., (2007); Benner et al., (2013) Lin et al., (2003); Sage et al., (2008); Sezaki et al., (2005); Thakar et al., (2005)

Alzheimer’s disease Cagliani et al., (2013)

Frangogiannis et al., Lange-Asschenfeldt et al., Inflammation ad (2005); (2002); Frolova et al., (2010); immune response Grimbert et al., (2006); Papageorgiou et al., Mustonen et al., (2012); Lopez-Dee et al., (2011); (2012); Pluskota et al., (2005) Manna and Frazier, (2003); Vanhoutte et al., (2013)

Martin-Manso et al., (2012); Miller et al., (2013); Mumby et al., (1984); Negoescu et al., (1995); Riessen et al., (1998); Streit et al., (2000); Vallejo et al., (2000); Vanhoutte et al., (2013); abkowitz etY al., (1993); Zhao et al., (2001)

Harada et al., (2003); Amy et al., (2013); Kang et al., (2006); Hawighorst et al., (2001); Bredel et al., (2005); Kawakami et al., (2001); Kodama et al., (2001); Forster et al., (2011); Kawataki et al., (2000); Oshika et al., (1998); Korkola et al., (2003); Kodama et al., (2001); Dalla-Torre et al., (2006); Cancers Streit et al., (1999); Liang et al., (2005); Lindner et al., (1992); Ramaswamy et al., (2003) Sun et al., (2006); Ma et al., (2009); Liu et al., (2005); Tokunaga et al., (1999) Sorlie et al., (2001); Volpert et al., (2002); Sun et al., (2006); Wu et al., (2004); Turashvili et al., (2007) Yang et al., (2003)

CMV infection Cinatl et al., (1999)

Belmadani et al., (2007); Bhattacharyya et al., (2008); Dabir et al., (2008); Daniel et al., (2004); Poczatek et al., (2000); Raman et al., (2011); Diabetes and Raman et al., (2007); metabolic disorders Stenina, (2005); Stenina et al., (2003); Sweetwyne and Murphy- Ullrich, (2012); Wang et al., (2006); Wang et al., (2004); Wang et al., (2003b); Zhou et al., (2006) Chavez et al., (2012)

conditions and would suggest the functions performed by TSPs in Urry et al., 1998). TSP-2 may be important in palate development: in physiological and pathological processes. the earlier stages of palatogenesis, it was found throughout the extracellular matrix of shelf mesenchyme (Melnick et al., 2000), but later it was 5. Expression of TSPs in developing embryo restricted to the EM and bone of the maxilla. TSP-1 was also abundantly expressed in developing head mesenchyme, including the palate. TSPs are highly expressed in tissues of developing embryos of Developmental stage-dependent co-expression of TSP-1 and TSP-2 β various species. In some cases, the significance of high expression has was proposed to regulate TGF 1 activity. TSP-4 expression pattern sug- been examined and understood. For example, in drosophila, single TSP gests its role in the regulation of neurite outgrowth and differentiation of the fly is expressed in tendon during the formation of myotendinous of cartilage, tendons, and bone (Arber and Caroni, 1995; Tucker et al., junctions (Subramanian et al., 2007; Gilsohn and Volk, 2010). Its ex- 1995). In mouse embryo, TSP-5 is expressed in mesenchyme at day pression is induced by the tendon-specific transcription factor Stripe. E10, and by E19 it is clearly expressed in osteogenic and chondrogenic TSP accumulates in the myotendinous junction, and flies deficient in tissues (Di Cesare et al., 2000; Fang et al., 2000; Kipnes et al., 2003). Its TSP fail to form functional somatic musculature. expression in both the embryonic and adult tissues indicates its impor- Pluripotent human embryonic stem cells expressed and secreted tance in skeletal and cartilage development (Chen et al., 2004; Lin et al., TSP-1, and it was the highest expressed protein detected in expression 2005; Kong et al., 2010; Roman-Blas et al., 2010). fi profiling of these cells (LaFramboise et al., 2010) and potentiating cell Most of the studies that documented the expression pro les of TSPs division in cardiomyocytes. This observation suggested that TSP-1 in embryonic development are descriptive [in addition to the studies might participate in cardiogenesis and cardiac repair by stem cells. mentioned above (Corless et al., 1992; Laherty et al., 1992)andothers]. Similarly to the adult tissues, TSP expression is tightly regulated in em- Most molecular mechanisms of the regulation of the expression in the fi bryos and is associated with specific stages of embryonic development. developing embryos have never been addressed, and the signi cance Increased TSP-1 expression at mid-gestation was responsible for em- of the tightly timed distinct expression of each TSP is unclear in most fi bryonic lethality with pronounced heart defects and vascular abnormal- cases. However, similar to the investigation into expression pro les fi ities (Fouladkou et al., 2010). The expression of TSP-1 in mouse embryo and regulation of expression in adults, these studies suggest speci c fi brain was also time dependent with a peak at days 11 and 12 and down- distinct functions for ve TSPs in development. regulation at the later stages (Iruela-Arispe et al., 1993). The expression patterns of TSP-1 suggested a role in early CNS development (Adams 6. Therapeutic regulation of expression and Tucker, 2000). Differential functions in developing embryo are supported by the distinct profile of expression of TSP-1, TSP-2 and TSP-3: Traditionally, the regulation of expression was not the first choice in TSP-1 was observed transiently in the neural tube, head mesenchyme, seeking to target specific proteins for therapeutic purpose. Difficulties of and cardiac cushions and constitutively in the resident megakaryocytes delivery of the molecular regulators into the nucleus and lack of speci- of the liver and in circulating megakaryocytes. In contrast, high expres- ficity in the action of the expression activators and inhibitors prevented sion of TSP-2 was detected in the connective tissue: e.g., in pericardium, the practical use of expression regulation in most cases. In the case of pleura, perichondrium, periosteum, meninges, ligaments, reticular TSPs, the regulation of expression is not only poorly understood, but is dermis, and cartilage and bone precursors (Iruela-Arispe et al., 1993; also multi-staged and complex, in part due to large regulatory regions Tooney et al., 1998). TSP-2 was also expressed in blood vessels and of mRNA (TSP-1 and TSP-2) and numerous introns (all TSPs). skeletal myoblasts. mRNA of TSP-3 was restricted to brain, cartilage In an attempt to control the foreign body response and to prevent and lung. The overlapping expression of TSP-1 and TSP-2 was found the granuloma formation in response to implantation and to increase only in the kidney and the gut (Iruela-Arispe et al., 1993). The dynamic angiogenesis, plasmids for the expression of a sense and an antisense pattern of TSP expression in Xenopus and avian embryos also support cDNA for TSP-2 were delivered in a collagen solution, which was applied the distinct non-overlapping functions for TSPs (Tucker et al., 1997; to biomaterials implanted subcutaneously (Kyriakides et al., 2001).

TSP-2 sense cDNA reversed the foreign body response in Thbs2−/− References mice, and TSP-2 anti-sense cDNA reversed the granuloma formation in wild-type mice, demonstrating that manipulations with the ex- Adams, J.C., 2001. Thrombospondins: multifunctional regulators of cell interactions. Annu. Rev. Cell Dev. Biol. 17, 25–51. pression of TSPs may be a valuable approach to regulate angiogenesis Adams, J.C., Lawler, J., 2004. The thrombospondins. Int. J. Biochem. Cell Biol. 36, 961–968. and matrix deposition. This approach was successfully used to alter Adams, J.C., Lawler, J., 2011. The thrombospondins. Cold Spring Harb. Perspect. Biol. 3, the wound healing response in wild-type and Thbs2−/− mice using a009712. Adams, J.C., Tucker, R.P., 2000. The thrombospondin type 1 repeat (TSR) superfamily: an in vivo murine excisional wound healing model with the im- diverse proteins with related roles in neuronal development. Dev. 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Gene 193, 5–11. tively easily designed, produced and modified to stabilize the oligonu- Agah, A., Kyriakides, T.R., Lawler, J., Bornstein, P., 2002. The lack of thrombospondin-1 cleotide and to facilitate its better delivery into cells (Krutzfeldt et al., (TSP1) dictates the course of wound healing in double-TSP1/TSP2-null mice. Am. – 2005; Davis et al., 2006; Krutzfeldt et al., 2007; Scherr et al., 2007; J. Pathol. 161, 831 839. Agah, A., Kyriakides, T.R., Letrondo, N., Bjorkblom, B., Bornstein, P., 2004. Thrombospondin Horwich and Zamore, 2008; Stenvang and Kauppinen, 2008; Stenvang 2 levels are increased in aged mice: consequences for cutaneous wound healing and et al., 2008a, 2008b; van Rooij et al., 2008; Wu and Belasco, 2008; angiogenesis. Matrix Biol. 22, 539–547. Wang and Olson, 2009). The synthetic oligonucleotides are stable, and Almog, N., Henke, V., Flores, L., Hlatky, L., Kung, A.L., Wright, R.D., Berger, R., Hutchinson, L., Naumov, G.N., Bender, E., Akslen, L.A., Achilles, E.G., Folkman, J., 2006. Prolonged they are retained in tissues for weeks. Despite a large number of predict- dormancy of human liposarcoma is associated with impaired tumor angiogenesis. ed targets for each miRNA that can be found in public databases, the FASEB J. 20, 947–949. predictions based solely on the sequence match not necessarily become Amodeo, V., Bazan, V., Fanale, D., Insalaco, L., Caruso, S., Cicero, G., Bronte, G., Rolfo, C., Santini, D., Russo, A., 2013. Effects of anti-miR-182 on TSP-1 expression in human experimentally proven targets. The regulatory mechanisms mediated colon cancer cells: there is a sense in antisense? Expert Opin. Ther. Targets 17, by miRNA now appear to be more complex than simple no-nonsense 1249–1261. binding of miRNA to the target and degradation of the sequence- Amy, E.M., Song, S., Kutasovic, J.R., Reid, L.E., Valle, J.M., Vargas, A.C., Smart, C.E., Simpson, P.T., 2013. Thrombospondin-4 expression is activated during the stromal response to matched mRNA. Thus, if a mechanism operating through miRNA is invasive breast cancer. Virchows Arch. 463, 535–545. dissected, targeting of this mechanism may be specificandefficient. Arber, S., Caroni, P., 1995. Thrombospondin-4, an extracellular matrix protein expressed We have demonstrated that the antagonist of miR-467 efficient- in the developing and adult nervous system promotes neurite outgrowth. J. Cell – ly prevents hyperglycemia-induced cancer angiogenesis and tumor Biol. 131, 1083 1094. Axel, D.I., Frigge, A., Dittmann, J., Runge, H., Spyridopoulos, I., Riessen, R., Viebahn, R., growth in mouse models by targeting miR-467 interaction with 3′UTR Karsch, K.R., 2001. All-trans retinoic acid regulates proliferation, migration, differen- of TSP-1, relieving the translational silencing of TSP-1, and increasing tiation, and extracellular matrix turnover of human arterial smooth muscle cells. – TSP-1 production (Sanghamitra Bhattacharyya et al., 2012). As the func- Cardiovasc. Res. 49, 851 862. Bauer, P.M., Bauer, E.M., Rogers, N.M., Yao, M., Feijoo-Cuaresma, M., Pilewski, J.M., tions of TSPs in different pathologies are better understood and the Champion, H.C., Zuckerbraun, B.S., Calzada, M.J., Isenberg, J.S., 2012. Activated CD47 miRNA regulation of TSPs is uncovered, targeting protein synthesis by promotes pulmonary arterial hypertension through targeting caveolin-1. Cardiovasc. miRNA may prove to be an efficient way to prevent or restore the pro- Res. 93, 682–693. Bein, K., Ware, J.A., Simons, M., 1998. Myb-dependent regulation of thrombospondin 2 duction of a protein. In the case of miR-467, the pathway is signal- and expression. Role of mRNA stability. J. Biol. Chem. 273, 21423–21429. tissue-specific(Bhattacharyya et al., 2008; Sanghamitra Bhattacharyya Bein, K., Odell-Fiddler, E.T., Drinane, M., 2004. Role of TGF-beta1 and JNK signaling in et al., 2012). For TSPs that are normally tightly regulated and expressed capillary tube patterning. Am. J. Physiol. Cell Physiol. 287, C1012–C1022. fi Belmadani, S., Bernal, J., Wei, C.C., Pallero, M.A., Dell'italia, L., Murphy-Ullrich, J.E., Berecek, in large amounts only in speci c and limited conditions, regulation with K.H., 2007. A thrombospondin-1 antagonist of transforming growth factor-beta miRNAmayprovetobeanefficient and harmless therapeutic approach. activation blocks cardiomyopathy in rats with diabetes and elevated angiotensin II. The fact that all of the five knockout mice did not demonstrate an overt Am. J. Pathol. 171, 777–789. fi Benner, E.J., Luciano, D., Jo, R., Abdi, K., Paez-Gonzalez, P., Sheng, H., Warner, D.S., Liu, C., phenotype without challenge suggests that general non-speci cdown- Eroglu, C., Kuo, C.T., 2013. Protective astrogenesis from the SVZ niche after injury is regulation of a single or several TSPs may be safe, at least for a short controlled by Notch modulator Thbs4. Nature 497, 369–373. period of time. Bentley, A.A., Adams, J.C., 2010. The evolution of thrombospondins and their ligand- binding activities. Mol. Biol. Evol. 27, 2187–2197. Bhattacharyya, S., Marinic, T.E., Krukovets, I., Hoppe, G., Stenina, O.I., 2008. Cell type- 7. Conclusion specific post-transcriptional regulation of production of the potent antiangiogenic and proatherogenic protein thrombospondin-1 by high glucose. J. Biol. Chem. 283, – Regulation of TSP expression is not a well-studied area, with the 5699 5707. 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