Invoking the Power of Thrombospondins: Regulation of Thrombospondins Expression

Abstract

Increasing evidence suggests critical functions of thrombospondins (TSPs) in a variety of physiological and pathological processes. With the growing understanding of the importance of these matricellular proteins, the need to understand the mechanisms of regulation of their expression and potential approaches to modulate their levels is also increasing. The regulation of TSPs expression is multi-leveled, cell- and tissue-specific, and very precise. However, the knowledge of mechanisms modulating the levels of TSPs is fragmented and incomplete. This review discusses the known mechanisms of regulation of TSPs levels and the gaps in our knowledge that prevent us from developing strategies to modulate the expression of these physiologically important proteins.

Introduction

Thrombospondins (TSPs) are secreted extracellular proteins that belong to a group of matricellular proteins - proteins that are present in the extracellular matrix (ECM), but do not play a primary structural role as other ECM proteins do. Matricellular proteins, including TSPs, have diverse functions that integrate ECM and cells: they interact with a number of cell surface receptors and with a variety of ECM proteins (e.g., structural proteins such as collagens, proteases, growth factors, etc.). The revolutionary classification of matricellular proteins proposed by late Paul Bornstein (1, 2) has promoted a better appreciation of ECM and its role in important physiological and pathological processes, as well as understanding that cells do not function in isolation from their environment and ECM.

The human TSP protein family consists of five members (TSP-1, TSP-2, TSP-3, TSP-4, and TSP-5, or cartilage oligomeric matrix protein, COMP), which are divided in two subgroups based on their domain structure (3-5) (TSP-1 and TSP-2 belong to subgroup A, while TSP-3, TSP-4, and TSP-5 compose subgroup B). In lower organisms, TSPs are represented by a single protein, as in sponge (6), or by multiple family members, e.g., five proteins in sea anemone (7), that have a structure similar to human TSPs of subgroup B. The second subgroup A appears later in the evolutionary development, coinciding with the development of the vascular system (6), and develops further as a reflection of the development of cardiovascular and immune systems where this group plays an important role.

Important physiological functions of TSPs have been discovered in many organs and systems [reviewed in (8-11)]. Regulation of angiogenesis and cancer progression (12), regulation of inflammation (8, 13-16), modulation of immune response (17, 18), formation of myotendinous junctions (19), maintenance of the myocardium integrity and function (20-27), regulation of fibrosis (28), and synaptogenesis (29) are just a few examples of their roles in physiology and pathology. To participate in the critical physiological processes, TSPs have to be present in the right location at the right time. This review summarizes the published information about the regulation of the expression of TSPs.

1. Similarities and distinctions between TSP family members

All TSPs share a high degree of homology at the protein level, especially in their “signature domain” - the C-terminal half of the human protein that includes 3-4 EGF-like repeats [1 – 5 in other species (4)], the Ca²⁺-binding domains with up to 26 metal binding sites per protein subunit, and the globular C-terminal domain [reviewed in (4)]. Table 1 summarizes the extent of homology between five TSPs. However, apart from the protein coding regions of the TSP genes (THBS), there is no homology in their DNA sequence, and the regulatory parts of TSP genes, either the untranslated regions of mRNA (UTR) or the gene promoters, are distinctly different at the first glance (30). Search for common DNA sequence motifs using SCOPE motif finder (http://genie.dartmouth.edu/scope)(31, 32) and comparison of the positions of common DNA sequence motifs in the promoters of five TSPs reveals some similarities between TSP promoters and suggests that in some conditions two or more TSPs may be regulated by the same stimuli simultaneously (Fig.2). However, there are no experimental data to support the predicted similarity of the DNA motifs.

Table 1.

Homology of human TSP proteins [calculated using algorithm described in (216)].

	TSP-1	TSP-2	TSP-3	TSP-4
TSP-1
TSP-2	62.6
TSP-3	52.2	52.9
TSP-4	53.9	52.6	61
TSP-5	53.6	54.4	65.3	69.9

Figure 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 (33). 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, heart.

The five proteins are located on different chromosomes (human THBS1 on chromosome 15 and mouse Thbs1 on chromosome 2, human THBS2 on chromosome 6 and mouse Thbs2 on chromosome 17, human THBS3 on chromosome 1 and mouse Thbs3 on chromosome 3, human THBS4 on chromosome 5 and mouse Thbs4 on chromosome 13, human THBS5 on chromosome 19 and mouse Thbs5 on chromosome 8).

In most tissues, TSPs are expressed at a low level compared to other non-structural ECM proteins, e.g., SPARC, tenascin C and fibronectin (Fig.3)(33). Similar to TSPs, tenascin C and SPARC are representatives of the matricellular protein family. SPARC and fibronectin regulate collagen deposition and ECM assembly and support the interaction between cells and ECM, similar to TSPs. These proteins have similar functions in disease regulation: e.g., they support interaction of cancer cells with stromal cells.

Figure 3.

Promoter of TSP-1: regulatory regions and experimentally confirmed binding sites for transcription factors.

Emphasizing the potent effect of TSPs presence in the tissue and the need to tightly regulate their expression, there are rapid mechanisms upregulating TSPs at the transcriptional level (34-36) and mechanisms rapidly degrading mRNA or blocking its translation into a protein (37-40). The proteins appear to be unstable after they are secreted, stressing the importance of timely elimination of TSPs from the ECM and cell environment (41-43). In adult organisms, upregulation of TSPs is associated with specific stages of wound healing [e.g., (36, 44-48)] and tissue remodeling [e.g., (13, 24, 25, 49-52)], in which it can be either protective and beneficial or detrimental.

Distinct localization of TSPs in tissues suggests differential functions, despite the high homology between proteins and a number of shared cell surface receptors and binding partners. Even when two or more TSPs are found in the same tissue, they are localized to the different cell types or structures within the tissue. For example, both TSP-3 and TSP-5 are present in the atherosclerotic lesion of ApoE^−/− mice, but they are clearly produced by different cell types in the lesion (13). Similarly, in the wall of smaller blood vessels TSP-3, TSP-4, and TSP-5 not only are produced by different cell types, but also are deposited in ECM in distinct patterns and in distinct localization within the vessel (53). In tendon, both TSP-3 and TSP-4 are abundant, but arranged in fibers of different orientation, stressing the distinctions in their functions (53). When TSPs are expressed in the same structures at the same time [e.g., TSP-4 and TSP-5 in tendon (53)], it is still unclear why both are required and what differential properties of tissue they support.

Differences in the expression profiles of individual TSPs in cancers and other cells and tissues [e.g., (48, 54, 55)] suggest that TSPs have differential functions in pathological processes, despite shared homologous domains and ligands. Several gene expression studies demonstrated opposite profiles for TSP-1 and TSP-4 in brain (56-58) and breast (59-61) cancers. Examination of the datasets from these studies reveals that TSP-4 upregulation is accompanied by downregulation of TSP-1 in the tumor samples (www.oncomine.org), clearly indicating distinct, probably even opposite, functions for these two proteins. Unexpectedly, TSP-2, belonging to the subgroup A and sharing high homology with TSP-1, exhibits a profile similar to the TSP-4 profile – it is upregulated in both brain and breast cancers, with the exception of one data set (57). In some datasets TSP-2 was in top 1% of upregulated genes, together with TSP-4. These comparisons of the three TSPs profiles from the same datasets clearly demonstrate that the three TSPs play important distinct roles in cancer growth, and the regulation of their expression is an efficient mechanism to support these still poorly understood functions (with the exception of TSP-1, whose downreguation is known to support angiogenesis in tumors). TSP-5 was upregulated similar to TSP-4 in multiple datasets from breast cancer studies (www.oncomine.org), while TSP-3 data were inconsistent, and both down- and up-regulation were observed (62, 63).

The expression studies in human samples and in animal tissues clearly suggest distinct functions and roles in physiological and pathological processes for five TSPs. However, the molecular mechanisms responsible for the differences in TSPs functions still have to be addressed and confirmed by examining specific functions of all five TSPs and by studying their interactions with cells and ligands. Precise regulation of TSPs expression is especially important in a view of TSPs homology and a number of shared ligands.

2. Mechanisms of regulation of TSPs expression

Most of the information about the regulation of TSPs expression comes from descriptive studies that either used array type experiments to identify highly upregulated or downregulated genes in tissues of interest or specifically looked at the TSPs expression (in most cases expression of TSP-1), based on known functions of TSPs. However, a few reports address specific molecular mechanisms of regulation of TSPs expression in cells and tissues.

2.1. Transcriptional regulation

2.1.1. TSP-1

2.1.1A Positive regulation

The first analysis of TSP-1 promoter and identification of the serum response element was reported by Framson and Bornstein in 1993 (64). The identified serum response element is bi-partite and includes a distal element at the position −1280 and a proximal element with NF-Y binding site at the position −65 (Fig.3). Interestingly, the proximal element is different in the mouse TSP-1 promoter: it harbors Egr-1 binding site in the position of human NF-Y site and does not participate in serum response. The Egr-1 binding site maintained constitutive activity of the promoter acting in concert with the adjacent GC-rich region binding SP1 (65). The importance of the first intron for the efficient transcription of the gene was noted (66): the deletion of this region results in 4-fold decrease in the reporter production, suggesting a cis-acting positive element in the first intron.

AP-1 binding to TSP-1 gene promoter mediates the activation of the promoter in response to protein kinase C (PKC) activation in human hepatoma cells (67). However, a similar study of the effects of PKC activation in porcine aortic endothelial cells resulted in opposite conclusions: a specific region of the promoter was responsible for the downregulation of the promoter activity (68). The cell-specificity is a very common theme in the studies of TSPs expression. The cell-and tissue-specific differences in regulation reflect the multi-functional nature of these proteins and the importance of a very strictly localized and timely production and elimination of TSPs.

The cell-specificity of TSP-1 gene promoter regulation was reported in several studies. The activity of a promoter is determined not only by its sequence, but also by availability of target binding sites in a specific cell type, presence of transcription factors capable of binding the promoter, and, finally, cell-specific signals to activate the transcription factors or protein co-activators. The regulatory complex assembled on TSP-1 gene promoter is often cell-specific, and the promoter regions involved in regulation are also different depending on the cell type. For example, in response to high glucose, TSP-1 mRNA is upregulated in major cell types of the vascular wall – endothelial cells, smooth muscle cells and fibroblasts (35). However, the time of upregulation is distinctly different – in endothelial cells upregulation can be detected earlier than in smooth muscle cells. The analysis of the promoter revealed that in two cell types the promoter activity is regulated by different transcriptional protein complex. In endothelial cells, a proximal regulatory region of the promoter binding Aryl Hydrocarbon Receptor is sufficient to drive the transcription (34, 69), while in smooth muscle cells the same promoter region is interacting with a distal part of the promoter through the formation of a single protein complex between transcription factors binding to the proximal and to the distal regions (69, 70). The regulation is determined by a cell-type-specific profile of the expression and the activation of transcription factors.

Transcriptional upregulation of TSP-1 in response to high glucose is probably the most dissected pathway among all the pathways regulating TSP-1 expression. TSP-1 has been implicated in development of vascular complications of diabetes, and its mRNA was dramatically upregulated in hyperglycemia in various tissues and organs (28, 35, 37, 38, 69, 71-78). The initial observation of upregulation of TSP-1 in response to high glucose in mesangial cells (73) was later confirmed in multiple cell types and tissues. The precise transcriptional mechanisms appear to vary depending on the cell type, with several transcriptional factors and regulatory regions being constant independently of the cell type – e.g., upstream stimulatory factors USF (74) that are common mediators of glucose effects. Another transcription factor activated by high glucose and essential for TSP-1 gene promoter activation is Aryl Hydrocarbon Receptor, or AhR (34, 69). The activation of the AhR pathway may provide a mechanistic connection between glucose and hypoxia regulatory pathways (AhR is a ligand for HIF1β or Arnt, whose other well studied ligand is HIF1α, a transcription factor that mediates many effects of hypoxia in complexes with Arnt). AhR is a member of the Clock family of proteins and can form complexes 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 (79, 80) prompted an investigation of transcriptional regulation of TSP-1 gene by leptin, a hormone implicated in development of obesity and diabetes. Leptin induced TSP-1 at the transcriptional level through the by JAK2/ERK/JNK-dependent mechanism (78).

The promoter of TSP-1 gene has an active Egr-1 binding site. The promoter activity is upregulated by Egr-1-inducing stimuli (81). TSP-1 mRNA can be very rapidly (within 10-15 min) upregulated in response to injury, both in vivo and in vitro (35), and, although not investigated, this upregulation most probably requires Egr-1, one of the first-response transcription factors. Egr-1 is responsible for transcriptional upregulation of TSP-1 by thrombin, together with MYC, in a thrombin receptor PAR-1, G(i/o), G(q), EBOX/EGRF-signaling cascade (82).

TSP-1 is a potent anti-angiogenic protein, and most of the reports on its expression and transcriptional regulation are related to regulation of angiogenesis in various tissues and pathological conditions. It is difficult to judge whether the focus on its anti-angiogenic function is due to the fact that this is truly the main function of TSP-1, whether this is due to the current popularity of the angiogenesis topics, or whether this is a result of a lack of sufficient understanding and knowledge about other functions of TSP-1. TSP-1 inhibits angiogenesis even in the presence of pro-angiogenic factors (83-85).

CCAAT box binding CCAAT-binding factor (CBF) mediates the effect of Histone Deacetylase (HDAC) inhibitors in regulation of angiogenesis and tumor growth: acetylated CBF specifically binds to TSP-1 promoter, and its activity is increased by the binding of acetylated H3 (86).

Recently, the role of TSP-1 in inflammation has been actively studied and confirmed in several models (87). Transcriptional regulation of TSP-1 in chronic inflammation was addressed in inflammatory joint disease (87): the orphan receptor 4A2 (NR4A2) was responsible for the downregulation of the promoter activity, and the anti-tumor necrosis factor treatment, which decreases NR4A2, increased TSP-1 expression and suggested that TSP-1 plays a role in resolution of inflammation.

Many studies reported the stimuli regulating transcription without addressing specific transcriptional mechanisms and transcription factors involved.

For example, TGFβ1 and TGFβ2 stimulated TSP-1 and TSP-2 production in bovine adrenocortical cells, and the effect was blocked by the transcription inhibitors (88).

2.1.1B Negative regulation

TSP-1 gene promoter appears to have an inhibitory element between −300 and −750 bp positions, as was reported in two independent studies (34, 89). Shorter promoter deletion constructs are significantly more active, as are the longer constructs, both constitutively and in response to stimuli.

The promoter was found to be sensitive to treatment with nickel (90): nickel downregulated the activity of the promoter and decreased the expression of endogenous TSP-1 in cultured hamster embryo cells. Activating Transcription Factor-1 (ATF-1) was identified as a component of the protein complex that bound to a negative regulatory site in the mouse TSP-1 gene promoter (91). ATF-1 binding to the region between positions −1210 and −1123 of the promoter harboring a cAMP-responsive element (CRE) induced the repression of the promoter in response to hepatocyte growth factor (HGF) and played a key role in tumor progression triggered by HGF (92).

Further analyses of THBS1 promoter identified many active binding sites for transcription factors that tightly regulate TSP-1 expression at the transcriptional level. YY-1 binds to a site at the position −440 of the promoter, which results in an interaction that can be weakened by the increased c-Jun levels, suggesting that the interaction of YY- and c-Jun decreases the promoter activity (89).

The well-documented suppression of TSP-1 production in tumors (upregulating angiogenesis to promote tumor growth) includes transcriptional downregulation: e.g., the product of Wilms' tumor suppressor gene, WT1, binds to the −210 region of the TSP-1 gene promoter and represses THBS1 transcription (93), while tumor suppressor protein p53 activates TSP-1 promoter (94). Another mechanism used by cancers to prevent TSP-1 promoter activation is hypermethylation of the promoter that has been detected in a variety of cancers [e.g., (95-98)]. TSP-1 promoter is also a target for a transcriptional factor Id1 that is known to regulate tumor angiogenesis (99).

Importantly, transcriptional repression is a common mechanism regulating the expression of TSP-1 – it results in a rapid effect, because both the protein and mRNA of TSP-1 are unstable (69, 70, 78, 100, 101).

MicroRNA regulation is usually associated with the modulation of mRNA translation or stability. However, miR-182 was reported to regulate the transcription of TSP-1 gene in colon cancer by modulating the function of transcription factors Egr-1 and Sp-1 function and their binding to THBS1 promoter (102).

Both TSP-1 and TSP-2 were downregulated in Akt^−/− mice, and this downregulation was responsible for the increased angiogenesis in these mice. The TSP-2 promoter inactivation by dominant negative Akt suggested that the regulation is transcriptional, but the exact mechanism has not been addressed (103). Although the studies identifying the signals that lead to the transcriptional upregulation are informative in defining the stimuli regulating the expression of TSPs, without addressing the molecular mechanism and delineating the promoter elements involved in the regulation it is impossible to know whether the observed effects are direct or mediated by an autocrine mediator and whether the mechanism of the regulation in these examples is truly transcriptional.

2.1.2. TSP-2

The promoters of human and mouse TSP-2 genes were analyzed and compared in (30). Interestingly, tissue-specific differences in transcription sites were found, resulting in transcripts of different lengths depending on the tissue. This was the only report considering the features of THBS2 promoter. Putative transcription factor binding sites were analyzed, and comparison to THBS1 promoter did not reveal any similarities between two genes in the promoter regions.

2.1.3. Other TSPs

Dr. Bornstein's group reported on the promoter of THBS3 as well. Interaction between THBS3 gene and adjacent metaxin gene was revealed (104), and the active SP1-binding elements were identified in the common regulatory region of the two genes (105). Analysis of the transcription initiation sites revealed alternative transcripts (106). Since the last publication in 1999, there have been no additional reports on regulation of THBS3 transcription.

There is very little information about the transcriptional regulation of TSP-4 and TSP-5 genes, despite their reported importance in several physiological and pathological processes (13, 24-26, 29, 52, 107, 108). The promoter of TSP-5 (COMP) has been analyzed, and a region required for the activity in chondrocytes was identified within 375 bp of the translational start site, as well as enhancer elements between −1.0 kb and −1.7 kb (109).

2.2. Alternative splicing

With a large number of exons in TSPs genes (>20 in most TSPs), the possibility of alternative splicing and regulation of expression and functions by this mechanism is plausible. However, alternative splicing and its potential significance in regulation of TSPs has not been seriously addressed to date. Several publications reported alternative forms of TSP-2 and TSP-3 (106, 110). A production of a Lumbar-disc-herniation-associated form of TSP-2 is due to a polymorphism and the alternative splicing of TSP-2 that results in skipping exon 11 (111). Lack of exon 11 causes decreased interaction of TSP-2 with metalloproteinases. Alternative splicing of TSP-4 gene associated with change in cell motility was reported in cancer cells (112).

Multiple TSPs forms of different sizes are routinely observed (e.g., (35, 113)), but the origin of these forms has not been thoroughly explored, and the possibility of the alternative splicing has not been excluded.

2.3. Regulation of mRNA stability

As has been mentioned above, both TSP-1 mRNA and protein have a short half-life. The short half-life of TSP-1 mRNA is due, at least in part, to AU-rich elements in the 3’ untranslated regions (3'UTR) of the TSP-1 mRNA (100, 114). Unusually large regulatory 3’UTR of TSP-1 and TSP-2 mRNA suggest extensive post-transcriptional regulation at the level of mRNA (Table 2). The rest of TSPs have an average sized UTR; however, this does not exclude regulation of mRNA stability or translation. UTR of all TSPs have putative target binding sites for miRNAs. There is no information on regulation of TSP-3, TSP-4, and TSP-5 expression at the level of mRNA, but several reports described the regulation of TSP-1 and TSP-2 through the modulation of mRNA stability or translation.

Table 2.

Size of untranslated regions of mRNA of TSPs.

	5’UTR	3’UTR
TSP-1	179	2130
TSP-2	320	2061
TSP-3	21	234
TSP-4	191	156
TSP-5	36	161

TSP-1 mRNA was destabilized by activation of myc oncoprotein (39). Further analysis of this mechanism revealed that destabilization is a result of increased expression of miR-17-92 family that bind TSP-1 mRNA (115). The decrease in the levels of the members of this family of miRNA, miR-18/19, was associated with increased TSP-1 level in cardiomyocytes of aging hearts, identifying the aging process (116). p53-responsive miR-194 decreased the levels of mature TSP-1 mRNA despite the increased levels of the primary transcript, prevented the production of TSP-1 protein, and promoted angiogenesis in colon cancers (117), presumably, due to destabilization of TSP-1 mRNA.

Increased TSP-1 mRNA stability was detected in response to heat shock (118), and a region of TSP-1 3’UTR responsible for the increased stability was identified as 968 – 1258 from the stop codon. TGFβ also increased TSP-1 expression in cultured osteosarcoma cells by stabilizing mRNA via p38 MAPK signaling (119).

There are no mechanistic reports describing the effects on TSP-2 mRNA stability. Although there are examples of post-trancriptional regulation of TSP-2 [e.g., in (120) describing the effect of c-myb on TSP-2 expression via a post-transcriptional mechanism], the details of this regulation remain unknown.

2.4. Regulation of mRNA translation

Due to the complexity of translational mechanisms and lack of detailed knowledge of these mechanisms, as well as to the requirement of specific skills and experience for the experimental manipulations with RNA, investigation of translational regulation of TSPs has not been the most popular of research topics. The large size of TSPs mRNA and, especially, the long regulatory UTR of TSP-1 and TSP-2 may have discouraged this type of study. However, studies of translational regulation of TSPs may prove very rewarding: there are multiple independent observations that increased levels of mRNA do not guarantee increased production of the corresponding protein and that the increased production of a protein may not always reflect the increased levels of mRNA [e.g., (37, 38, 43)]. As more examples of such discordant levels of protein and mRNA accumulate, we will be forced to address the translational regulation of TSPs.

TSP-1 mRNA is associated with rich in adenines and uracils ARE binding protein HuR. HuR facilitates translation, and it has been co-precipitated with TSP-1 mRNA from the cultured MCF7 breast cancer cells (121). Another example of translational regulation of TSP-1 production is translational silencing of TSP-1 mRNA in response to high glucose (37, 38) that is tissue-specific and regulated by a cell-specific induction of miR-467 and its binding to TSP-1 3’UTR.

2.5. Post-translational regulation – secretion

TSPs are secreted extracellular matrix proteins. Although some of them, e.g., TSP-1, TSP-4 and TSP-5, perform important functions inside the secretory pathways (25, 122-124), most of the widely known functions rely on the presence of TSPs outside the cell.

The regulation of secretion of pre-made protein is a very fast and a very efficient way to regulate the protein availability, and this type of regulation is used by the cells to control the extracellular TSPs levels. TSP-1 can be retained inside the cells, and the secretion depends on the Ca2+ regulation: TSP-1 retention inside the cells can be induced by a calcium chelator and is regulated in renal cell carcinoma cells by TRPC4 calcium channel expression (125). TSP-1 is a major component of platelets alpha granules: pre-synthetised protein is released upon platelets activation (126, 127). Macrophages also regulate TSP-1 secretion: the secretion is induced by LPS, and the threshold for the level of secretion is regulated by the environmental signals, including the mediators of the adaptive immune system, Th1 and Th2 cytokines (128). TSP-1 secretion depends on cell density in culture: lower density results in higher secretion of TSP-1 by fibroblasts, endothelial and smooth muscle cells without change in protein synthesis (129).

Both TSP-4 and TSP-5 appear to have additional intracellular functions in the secretory pathways: TSP-5 can associate with its co-secreted extracellular ligands and ER chaperones (122-124), and TSP-4 bind a transcriptional factor Aft6α promoting its translocation to the nucleus (25). The latter function is shared with TSP-1 and is thought to be a common feature of TSPs in protecting and augmenting ER function (25).

3. Descriptive studies of TSPs expression

More than 2,400 publications address the expression of TSPs in different tissues and various conditions. The findings clearly indicate the importance of the altered expression in physiology and pathology and stress the need to develop understanding of the mechanisms that control the expression. Most of the information about the expression of TSPs comes from descriptive studies, in which the mechanisms of regulation have not been addressed. This information is valuable in identifying 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 levels have been assessed. Whenever only the mRNA levels were evaluated, there is always a possibility that they do not translate into protein production or that the produced protein is not secreted, as seems to be a frequent occurrence with TSPs.

Although an overview of conditions resulting in altered expression of TSPs is not the goal of this article, several major themes in regulation of TSPs are very prominent and must be mentioned: growth and remodeling of tissues, altered angiogenesis and cancer, ischemia and reperfusion, activation of TGFβ, inflammation and immune response, aging, and embryonic development (Table 3). Any of these pathological or physiological situations consistently result in or are caused by the change in the levels of one or several TSPs. As usual, TSP-1 expression has been better studied than the expression of the other four TSPs, although many recent reports repeatedly find the associations of TSP-4 expression and pathological changes.

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 and the downregulated expression has been reported.

	TSP-1	TSP-2	TSP-3	TSP-4	TSP-5 (COMP)
Heart hypertrophy	Wang et al., 2003a	Schroen et al., 2004; Swinnen et al., 2009; van Almen et al., 2011a		Cingolani et al., 2011; Frolova et al., 2012; Gabrielsen et al., 2007; Lynch et al., 2012; Melenovsky et al., 2011; Mustonen et al., 2008; Mustonen et al., 2010; Rysa et al., 2005
Atherosclerosis	Frolova et al., 2010; Moura et al., 2008; Reed et al., 1995; Riessen et al., 1998	Frolova et al., 2012; Pohjolainen et al., 2012	Frolova et al., 2010	Frolova et al., 2010	Frolova et al., 2010
Fibrosis	Sweetwyne and Murphy-Ullrich, 2012	Pohjolainen et al., 2012; Reinecke et al., 2013
Arterial injury	Chen et al., 1999; Miano et al., 1993; Raugi et al., 1990; Roth et al., 1998; Sajid et al., 2001; Stenina et al., 2003
Ischemia, reperfusion	Favier et al., 2005; Frangogiannis et al., 2005; Kaiser et al., 2013; Lario et al., 2007; Lin et al., 2003; Sage et al., 2008; Sezaki et al., 2005; Thakar et al., 2005			Benner et al., 2013
Alzheimers disease				Cagliani et al., 2013
Inflammation ad immune response	Frangogiannis et al., 2005; Grimbert et al., 2006; Lopez-Dee et al., 2011; Manna and Frazier, 2003; 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; Yabkowitz et al., 1993; Zhao et al., 2001	Lange-Asschenfeldt et al., 2002; Papageorgiou et al., 2012; Vanhoutte et al., 2013		Frolova et al., 2010; Mustonen et al., 2012; Pluskota et al., 2005
Cancers	Harada et al., 2003; Kang et al., 2006; Kawakami et al., 2001; Kawataki et al., 2000; Kodama et al., 2001; Lindner et al., 1992; Liu et al., 2005; Volpert et al., 2002; Wu et al., 2004; Yang et al., 2003	Hawighorst et al., 2001; Kodama et al., 2001; Oshika et al., 1998; Streit et al., 1999; Sun et al., 2006; Tokunaga et al., 1999	Dalla-Torre et al., 2006; Ramaswamy et al., 2003	Amy et al., 2013; Bredel et al., 2005; Forster et al., 2011; Korkola et al., 2003; Liang et al., 2005; Ma et al., 2009; Sorlie et al., 2001; Sun et al., 2006; Turashvili et al., 2007
CMV infection	Cinatl et al., 1999
Diabetes and metabolic disorders	Belmadani et al., 2007; Bhattacharyya et al., 2008; Dabir et al., 2008; Daniel et al., 2004; Poczatek et al., 2000; Raman et al., 2011; Raman et al., 2007; 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
Pulmonary arterial hypertension	Bauer et al., 2012; Maloney et al., 2012
Wound healing	Agah et al., 2002; Agah et al., 2004; Bornstein et al., 2004; DiPietro et al., 1996; Raugi et al., 1987; Reed et al., 1993; Streit et al., 2000	Agah et al., 2002; Agah et al., 2004; Bornstein et al., 2004; Kyriakides et al., 1999
Skeletal and joints disorders					Posey and Hecht, 2008
Aging	Cai et al., 2012; Frazier et al., 2011; Starr et al., 2013; van Almen et al., 2011b	Agah et al., 2004; Swinnen et al., 2009		Cingolani et al., 2011
Embryonic development	Adams and Tucker, 2000; Di Cesare et al., 2000; Fouladkou et al., 2010; Iruela-Arispe et al., 1993; LaFramboise et al., 2010; Tucker et al., 1997; Urry et al., 1998	Adams and Tucker, 2000; Iruela-Arispe et al., 1993	Tucker et al., 1997; Urry et al., 1998	Arber and Caroni, 1995; Tucker et al., 1995	Chen et al., 2004; Di Cesare et al., 2000; Fang et al., 2000; Kipnes et al., 2003; Kong et al., 2010; Lin et al., 2005; Roman-Blas et al., 2010

Altered expression of TSPs during the growth and remodeling of tissues is associated either with the need for angiogenesis, matrix remodeling, or the direct stimulation of cell growth. TSP-4 is upregulated in the mouse heart in response to pressure overload and in human hypertrophied hearts. It has a protective function regulating cardyomyocyte function and production of extracellular matrix (24-26, 52, 130-132). TSP-4 is expressed in atherosclerotic lesions and promotes atherosclerotic process by attracting and retaining macrophages in the lesion (13). Thbs4 is responsive to the ischemic injury in the brain cortex and plays an important role in post-injury astrogenesis (133). THBS4 expression increases with age in the brain gray matter and may be associated with Alzheimers disease and altered inflammatory response (134). Increased TSP-1 expression is also often found in tissue growth and remodeling, e.g., in embryo fibroblasts in response to c-Jun stimulation (135). Increased levels of TSP-1 and TSP-2 have been reported in remodeling large arteries in atherosclerotic lesions and after injury (13, 35, 51, 80).

Inflammation and immunity is an emerging field in TSP studies (16, 136-145). Although functions of TSPs in immune response and inflammation are not completely understood, these proteins are clearly important for the adequate immune response and the resolution of the inflammation.

Multiple studies documented changed expression of TSP-1 and TSP-2 associated with altered angiogenesis and cancer growth. Decreased TSP-1 and/or TSP-2 expression correlates with increased vascularity in non-small cell lung cancers (146), colon cancer (147, 148), invasive cervical cancer (149, 150), glioma cells (151), in cytomegalovirus (CMV) infection (152), and many other situations where angiogenesis is increased. Interestingly, the expression of group B thrombospodins (TSP-3, TSP-4 and TSP-5) increases in cancers. Increased levels of TSP-3 were associated with poor prognosis in osteosarcoma patients (153). TSP-4 expression was increased in invasive breast cancer and was suggested to facilitate the invasion of tumor cells (154). It was differentially expressed in lobular versus ductal breast tumors (155). THBS4 was found to be a powerful marker of diffuse-type gastric adenocarcinomas: its expression in fibroblasts was stimulated by tumor cells (156). Based on the existing data, THBS4 expression is a selective marker for specific cancers, although the regulation and the significance of the increased levels have not been explored.

Many stimuli able to reduce TSP-1 levels have been identified: e.g., all-trans-retinoid acid in smooth muscle cells (157); Hepatocyte growth factor/scatter factor that mediates angiogenesis through positive VEGF and negative TSP-1 regulation (158); shear stress (159), and hypoxia (160). Increase in TSP-1 levels is often detected in association with decreased angiogenesis and ischemia, e.g., in chronic leg ischemia (161). Increased levels of TSP-1 mRNA were detected upon stimulation with dexamethasone (162); progesterone (163); high glucose (35, 73), thrombin and angiotensin II (164). TSP-2 expression is down regulated by Cyp1B1, and this regulation is important for a proper capillary morphogenesis in a model of retinopathy of prematurity (a neovascular response during oxygen-induced ischemic retinopathy)(165). The activity of endothelial nitric oxide synthase (eNOS) negatively correlated with TSP-2 levels: deficiency in eNOS resulted in increased levels of TSP-2 and decreased angiogenesis in a mouse model, and NO repressed the promoter of TSP-2 (166). However, the precise transcriptional mechanisms were not dissected in these studies.

The extent of angiogenesis after ischemia and reperfusion (IR) correlates with the successful repair process in tissues and with the patient survival. Elevation of TSP-1 levels was detected after IR of brain, myocardium, lung, and kidney (139, 167-170). While the expression of TSP-2 was elevated at the peak of angiogenesis (2 weeks after reperfusion), the expression of TSP-1 was dramatically elevated early after the event and did not seem to represent a response to the angiogenic process. Although it is unclear what the role of TSP-1 is in tissues just hours after IR, it appears to have a protective effect in heart: e.g., myocardial infarction (MI) patients with a higher levels of TSP-1 in the platelet-poor plasma had a better outcome and fewer adverse cardiac events within 14 months after MI (171). In animals models of MI, TSP-1 mRNA was induced only 1 hour of ischemia and was elevated 3-7 days after reperfusion. TSP-1 was localized to the infarction border zone and reduced inflammation and granulation tissue formation protecting non-infarcted myocardium from fibrosis (168, 171). Interestingly, in kidneys increased expression of TSP-1 early after IR (only 3 – 12 hours, returning to the base line by 48 hours) mediated the injury, and Thbs1^−/− mice showed significant protection from the damage after IR injury and from the renal failure (172). Clearly, the function and the effect of the altered expression of TSPs depend on the injury mechanism and the specific cellular events characteristic for a tissue.

TSP-1 is a major activator of TGFβ1, and there is a reciprocal feedback mechanism maintaining and amplifying the circuit: TGFβ was identified as TSP-1 expression regulator increasing the production of TSP-1 in multiple studies (162, 173). As a major activator of TGFβ1 (174), TSP-1 is often expressed in tissues producing TGFβ1, e.g., in cancers with high TGFβ1 levels (175) and in embryonic development (176). TSP-1 gene mutation was identified in a family with the familial pulmonary arterial hypertension (PAH)(177). The ability of the mutant Asp362Asn TSP-1 to activate TGFβ1 was reduced to ½ of the activity of the wild-type TSP-1. TSP-1 contributes to the development of PAH in more than one way: the levels of TSP-1 are increased in PAH patients and in animal models of PAH, and the interaction of TSP-1 with its receptor CD47 disrupts the constitutive interaction between CD47 and Caveolin-1, causing increased eNOS-dependent superoxide production and oxidative stress (178).

Levels of TSPs increase in multiple tissues with age (21, 116, 179-182). The significance of this increase in TSPs production is still unclear, although in some tissues it appears to be beneficial (21, 26, 179, 180). In others, TSPs are associated with aging-related pathological changes (27, 116, 182, 183).

While the significance of the altered expression of TSP-1 and TSP-2 is well understood in most situations, and specific consequences are expected (e.g., altered angiogenesis, matrix remodeling, TGFβ activation), we don't have a clear understanding of the significance of altered expression of group B TSPs in the majority of cases. Studies of the signals and molecular pathways regulating their expression would greatly increase our understanding of TSPs associations with specific pathological conditions and would suggest the functions performed by TSPs in physiological and pathological processes.

4. Expression of TSPs in developing embyo

TSPs are highly expressed in tissues of developing embryos of various species. In some cases, the significance of high expression has been examined and understood. For example, in drosophila, single TSP of the fly is expressed in tendon during the formation of myotendinous junctions (19, 184). Its expression is induced by the tendon-specific transcription factor Stripe. TSP accumulates in the myotendinous junction, and flies deficient in TSP fail to form functional somatic musculature.

Pluripotent human embryonic stem cells expressed and secreted TSP-1, and it was the highest expressed protein detected in expression profiling of these cells (185) and potentiating cell division in cardiomyocytes. This observation suggested that TSP-1 might participate in cardiogenesis and cardiac repair by stem cells. Similarly to the adult tissues, TSP expression is tightly regulated in embryos and is associated with specific stages of embryonic development. Increased TSP-1 expression at mid gestation was responsible for embryonic lethality with pronounced heart defects and vascular abnormalities (186). The expression of TSP-1 in mouse embryo brain was also time dependent with a peak at days 11 and 12 and down-regulation at the later stages (187). The expression patterns of TSP-1 suggested a role in early CNS development (188). Differential functions in developing embryo are supported by the distinct profile of expression of TSP-1, TSP-2 and TSP-3: TSP-1 was observed transiently in the neural tube, head mesenchyme, and cardiac cushions and constitutively in the resident megakaryocytes of the liver and in circulating megakaryocytes. In contrast, high expression of TSP-2 was detected in the connective tissue: e.g., in pericardium, pleura, perichondrium, periosteum, meninges, ligaments, reticular dermis, and cartilage and bone precursors (187, 189). TSP-2 was also expressed in blood vessels and skeletal myoblasts. mRNA of TSP-3 was restricted to brain, cartilage and lung. The overlapping expression of TSP-1 and TSP-2 was found only in the kidney and the gut (187). The dynamic pattern of TSPs expression in Xenopus and avian embryos also support the distinct non-overlapping functions for TSPs (190, 191). TSP-2 may be important in palate development: in the earlier stages of palatogenesis, it was found throughout the extracellular matrix of shelf mesenchyme (176), but later it was restricted to the EM and bone of the maxilla. TSP-1 was also abundantly expressed in developing head mesenchyme, including the palate. Developmental stage – dependent co-expression of TSP-1 and TSP-2 was proposed to regulate TGFβ1 activity. TSP-4 expression pattern suggests its role in regulation of neurite outgrowth and differentiation of cartilage, tendons, and bone (192, 193). In mouse embryo, TSP-5 is expressed in mesenchyme at the day E10, and by E19 it is clearly expressed in osteogenic and chondrogenic tissues (194-196). Its expression in both the embryonic and the adult tissues indicates its importance in skeletal and cartilage development (197-200).

Most of the studies that documented the expression profiles of TSPs in embryonic development are descriptive [in addition to the studies mentioned above (201, 202) and others]. Most molecular mechanisms of the regulation of the expression in the developing embryos have never been addressed, and the significance of the tightly timed distinct expression of each TSP is unclear in most cases. However, similar to the investigation into expression profiles and regulation of expression in adults, these studies suggest specific distinct functions for five TSPs in development.

5. Therapeutic regulation of expression

Traditionally, the regulation of expression was not the first choice in seeking to target specific proteins for therapeutic purpose. Difficulties of delivery of the molecular regulators into the nucleus and lack of specificity in the action of the expression activators and inhibitors prevented the practical use of expression regulation in most cases. In the case of TSPs, the regulation of expression is not only poorly understood, but is also multi-staged and complex, in part due to large regulatory regions of mRNA (TSP-1 and TSP-2) and numerous introns (all TSPs).

In an attempt to control the foreign body response and to prevent the granuloma formation in response to implantation and to increase angiogenesis, plasmids for the expression of a sense and an antisense cDNA for TSP-2 were delivered in a collagen solution, which was applied to biomaterials implanted subcutaleously (203). TSP-2 sense cDNA reversed the foreign body response in Thbs2^−/− mice, and TSP-2 anti-sense cDNA reversed the granuloma formation in wild-type mice, demonstrating that manipulations with the expression of TSPs may be a valuable approach to regulate angiogenesis and matrix deposition. This approach was successfully used to alter the wound healing response in wild-type and Thbs2^−/− mice using an in vivo murine excisional wound healing model with the improved polymer facilitating the delivery and release of the plasmid and increasing the efficiency of cell transfection in the healing wound (204).

The discovery and the rapid progression of our knowledge of miRNA rekindled the interest in therapeutic regulation of expression. Synthetic miRNA and their sequence-specific antisense antagonists can be relatively easily designed, produced and modified to stabilize the oligonucleotide and to facilitate its better delivery into cells (205-215). The synthetic oligonucleotides are stable, and they are retained in tissues for weeks. Despite a large number of predicted targets for each miRNA that can be found in public databases, the predictions based solely on the sequence match not necessarily become experimentally proven targets. The regulatory mechanisms mediated by miRNA now appear to be more complex than simple no-nonsense binding of miRNA to the target and degradation of the sequence-matched mRNA. Thus, if a mechanism operating through miRNA is dissected, targeting of this mechanism may be specific and efficient.

We have demonstrated that the antagonist of miR-467 efficiently prevents hyperglycemia-induced cancer angiogenesis and tumor growth in mouse models by targeting miR-467 interaction with 3’UTR of TSP-1, relieving the translational silencing of TSP-1, and increasing TSP-1 production (38). As the functions of TSPs in different pathologies are better understood and the miRNA regulation of TSPs is uncovered, targeting protein synthesis by miRNA may prove to be an efficient way to prevent or restore the production of a protein. In the case of miR-467, the pathway is signal- and tissue-specific (37, 38). For TSPs that are normally tightly regulated and expressed in large amounts only in specific and limited conditions, regulation with miRNA may prove to be an efficient and harmless therapeutic approach. The fact that all of the five knockout mice did not demonstrate an overt phenotype without challenge suggests that general non-specific downregulation of a single or several TSPs may be safe, at least for a short period of time.

6. Conclusion

Regulation of TSPs expression is not a well-studied area, with the exception of TSP-1 that attracted more attention, mostly due to the profound changes in its expression during tumor development and its potent effect on angiogenesis. The existing information suggests that the expression of TSPs is regulated at multiple molecular levels from transcriptional regulation and splicing to regulation of protein secretion. Furthermore, we learned that the regulation of expression is often not only cell- and tissue-specific, but also multi-leveled: simultaneous engagement of several levels of regulation results in discordant levels of mRNA and protein. Although investigation of different levels of regulation may appear tedious and complex, the detailed knowledge of complementary molecular mechanisms may eventually prove useful in developing effective, fast and specific therapeutic intervention.

Highlights.

1. Thrombospondins (TSPs) are highly expressed in embryonic development, tissue injury and remodeling, inflammation and immune response, and aging.

2. Expression patterns suggest distinct specific functions for each TSP.

3. Expression of TSPs is tightly regulated at several molecular levels: transcription, RNA processing and translation, protein stability and secretion.

4. Experimental regulation of TSPs expression modulates physiological and pathological processes dependent on TSPs.

Figure 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) (31, 32). Motifs positioned similarly in the promoters of two or more TSPs are shown in the figure (6 out of 20 most significant matches).

Abbreviations

AhR

Aryl (aromatic) hydrocarbon receptor

ARE

Adenylate-uridylate-rich element

ATF-1

Activating Transcription Factor-1

CBF

CCAAT-binding factor

COMP

Cartilage oligomeric matrix protein

Cyp1B 1

Cytochrome P450 1B1

EBOX

Enhancer box

ECM

Extracellular matrix

Egr-1

Early growth response protein 1

EGRF

Early growth response factor 1

eNOS

Endothelial nitric oxide synthase

ER

Endoplasmic reticulum

H3

Histone 3

HDAC

Histone Deacetylase

HGF

Hepatocyte growth factor

HuR

Human antigen R

IR

Ischemia / reperfusion

MAPK

Mitogen-activated protein kinases

MI

Myocardial infarction

MYC

Myelocytomatosis oncogene

NO

Nitric oxide

NR4A2

Nuclear receptor subfamily 4, group A, member 2

PAH

Pulmonary arterial hypertension

PAR-1

Protease-activated receptor-1

SPAR C

Secreted protein acidic and rich in cysteine

TGFβ

Transforming growth factor beta

TRPC 4

Short transient receptor potential channel 4

TSP

Thrombospondin

UTR

Untranslated region

WT1

Wilms' tumor suppressor

YY-1

Yin Yang 1