Search

Connective tissue fibrosis is the final common pathogenic process for almost all forms of chronic tissue injury. Whether caused by vascular dysfunction, inflammation, metabolic injury, trauma, or environmental agents, once initiated the fibrogenic process results in the progressive replacement of the normal tissue architecture with fibrotic lesions that eventually lead to organ compromise and failure. Fibrosis can be considered as a dysregulation in the normal tissue repair mechanism, resulting in severe tissue scarring. Fibrosis appears to be a consequence of linked processes, including the proliferation of resident fibroblast cell types, the increased production and deposition of extracellular matrix components, and the transition of fibroblasts into cells exhibiting a myofibroblast phenotype. Although transforming growth factor-beta (TGF beta) has long been regarded as a pivotal growth factor in the formation and maintenance of connective tissues and as a major driving influence in many progressive fibrotic diseases, attention has focused recently on the role of connective tissue growth factor (CTGF) in fibrosis. CTGF is selectively and rapidly induced in mesenchymally derived cells by the action of TGF beta. CTGF expression is increased in many fibrosing diseases. In addition, increasing evidence from in vivo and in vitro models of tissue remodeling and fibrosis suggest that CTGF may represent a downstream effector molecule of the profibrotic activities of TGF beta in the maintenance and repair of connective tissues and within fibrotic disease settings.

There is no therapy for chronic fibroproliferative diseases, in spite of the fact that current health statistics suggest that these (which include cardiovascular disease, pulmonary fibrosis, diabetic nephropathy, liver cirrhosis and systemic sclerosis) have been estimated to cause approximately 45% of the deaths in the developed world. Recently, many studies have shown that mitogen activated protein kinases (MAPKs) are activated in response to fibrogenic agents and contribute to the formation and function of the myofibroblast, the critical cell type responsible for excessive scarring. A recent report by Madala and colleagues (Am J Respir Cell Mol Biol, 2011) has provided a proof-of-concept study showing that the specific MEK inhibitor ARRY-142886 (ARRY) can both suppress the progression of fibrosis and reverse an animal model of lung fibrosis. Thus MEK inhibition could be a valuable method to treat lung fibrosis.

Approximately 45 people attended the recent ICCNS-sponsored meeting in Vancouver. The meeting commenced the evening of September 24, 2011 at the Steamworks Brewpub located on the Vancouver waterfront, with the lights from the Northshore mountains as a backdrop. People were impressed with the beer and wine selection, as well as the quality west-coast food. We were grateful to Springer for sponsoring this welcome dinner. After a hearty breakfast, the meeting officially started at the Coast Coal Harbour hotel with a session sponsored by the Canadian Institutes of Health Research. Unfortunately, Bernard Perbal (Paris, France) could not attend the meeting due to his move from Paris, nonetheless David Brigstock (Columbus, USA) stepped in at the last minute to provide an excellent overview of the structure and function of the CCN proteins and of the potential use of CCN2 antagonists in liver fibrosis. To continue the theme, Stephen Twigg (Sydney, Australia) and Enrique Brandan (Santiago, Chile) provided strong evidence that CCN2 was essential for controlling the intensity of fibrosis progression in models of diabetes-related non-alcoholic steatohepatitis and skeletal muscular dystrophy, respectively. Chris Overall (Vancouver, Canada) reminded us that the CCN proteins are highly modular, and that matrix metalloprotenases play a key role in modifying the activity of individual CCN molecules and that the function of individual fragments may be quite different. Karen Lyons (Los Angeles, USA) provided strong evidence that CCN1 and CCN2 may be somewhat similar functionally, and suggested that the dosing of these molecules may be important in vivo; deleting CCN1 and CCN2 together had more of an effect on bone development than deleting either protein alone. Thus drugs that target both CCN1 and CCN2 in vivo might have quite different effects than targeting one molecule alone. After an Italian-themed lunch, the Young Investigator session, sponsored by the Scleroderma Society, started. In this session, Alyssa Charrier (Columbus, USA) and Rohan Samarakoon (Albany, USA) both discussed the link between CCN2 expression and fibrosis, in particular alcohol-induced pancreatic injury and vascular smooth muscle cells, respectively. Taken together, Danilo Janune (Okayama, Japan) and Takashi Nishida (Okayama, Japan) indicated the yin/yang relationship between CCN3 and CCN2 regarding chondrocyte differentiation and/or proliferation. Alex Lambi (Philadelphia, USA) carefully described a heretofore unappreciated phenotype of the whole-body CCN2 knockout mice; namely that these mice possessed cranial defects consistent with impaired intramembranous and endochondral ossification. Finally, the essential role of matricellular proteins in tissue repair was further emphasized by Chris Elliott (London, Canada) who used the periostin knockout mouse to show that periostin was required for tissue repair, by correlating the role periostin on myofibroblast differentiation to matrix stiffness. After an afternoon poster session in which Vancouver Chinatown-themed refreshments were served, Brahim Chaquor (Brooklyn, USA) discussed how CCN1 might be used to treat vascular diseases of the eye. CCN1 expression is reduced in response to hyperoxia caused by neovascularization in retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Lester Lau (Chicago, USA) continued with CCN1 theme, and discussed recent data indicating that the CCN1/integrin α6β1 is important for controlling the kinetics of wound closure (and presumably fibrosis) by altering the accumulation of reactive oxygen species. Hannu Larjava (Vancouver, Canada) and Shoukat Dedhar (Vancouver, Canada) closed the session by discussing the importance of integrin-mediated adhesion to cell physiology by discussing how, in principle, inhibiting integrin β6 or integrin linked kinase might be used to control fibrosis or cancer. The meeting then adjourned until the morning of the 26th. In an interesting morning session, we were reminded by Lynne Postovit (London, Canada) that the surrounding matrix instructs cells to respond to signals; for example, the matrix produced by stem cells or cancer cells are distinct, and can program one cell type to behave like another cell type. Fabio Rossi (Vancouver, Canada) provided fascinating data involving the role of cell adhesion proteins in satellite cell activation during muscle generation. Andrei Thomas-Tikhonenko (Philadelphia, USA) discussed how the p53-responsive miR-194 and inhibits thrombospondin-1 and thus promotes angiogenesis. Ruth Lupu (Rochester, USA) and Chiayeng Wang (Chicago, USA) provided evidence suggesting that CCN1 promotes breast cancer, and that CCN3, by being a target of the a paired-domain-specific PAX3-FKHR transcription factor, promotes survival and motility in alveolar rhabdomyosarcoma cells. Shushanta Banerjee (Kansas City, USA) showed, on the other hand, that CCN5 is anti-oncogenic as CCN5 suppresses twist and then mir-10b, whose expression is increased in metastatic breast cancer cells and elevated cell migration through the suppression of the homeobox D10 (HOXD10) tumor suppressor. The gala dinner, sponsored by the ICCNS, was held in the evening at the extremely vibrant Joe Fortes Restaurant, where delegates dined on locally sourced oysters and seafood. The final session, on the morning of the 27th, focused on translational research and was sponsored by Actelion. Ron van Noorden (Amsterdam, Netherlands) pointed out that anti-VEGF therapies might not be appropriate to treat diabetic retinopathy given that an elevated CCN2/VEGF ratio correlates with increased fibrosis. Thus dual anti-VEGF and anti-CCN2 therapies need to be considered for this disorder. Chris Denton (London, UK) and Richard Stratton (London, UK) nicely described the autoimmune connective tissue disease scleroderma, and presented data showing that, in spite of much effort, there is no treatment for the fibrosis in scleroderma. Multiple drugs are being considered in clinical trials for scleroderma, but CCN2 is certainly a marker of fibrosis in this disease and also mediate the ability of scleroderma keratinocytes to promote fibroblast activation in this disease. Satoshi Kubota (Okayama, Japan) provided a highly amusing talk, in which he linked CCN2 expression to nicotine-induced remodeling of periodontal tissues. Finally, Roel Goldschmeding (Utrecht, Netherlands) summed up the meeting by providing exciting data suggesting that anti-CCN2 therapies may be useful to reverse the fibrosis seen in patients with diabetes. Overall, data was provided that strongly suggested that the CCN proteins, through their ability to modify cellular microenvironments and hence adhesive signaling, appear to be excellent targets for drug intervention in both cancers and connective tissue diseases.

Scleroderma (systemic sclerosis, SSc) is an autoimmune disease of unknown etiology characterized by organ fibrosis. There is no therapy for SSc. However, a recent body of evidence strongly implicates endothelin-1 (ET-1) in the pathogenesis of SSc. ET-1 is found in abundance in SSc patients. ET-1 directly induces fibrogenic effects in vitro, and is required for the ability of TGFβ to induce fibrogenic effects both in vitro and in vivo. Moreover, endothelin receptor antagonism reverses key features of the persistent fibrotic phenotype of fibroblasts isolated from lesions of SSc patients. However, clinically, endothelin receptor antagonism alone has had mixed results. This minireview summarizes these observations.

Members of CCN family of matricellular proteins are being increasingly recognized by the translational research community as representing excellent targets for drug intervention. Although much effort has been expended in outlining the mechanisms involved in pancreatic carcinogenesis, the precise molecular pathways involved remain incompletely understood, and appropriate targets for drug intervention remain elusive. A recent exciting report by Haque and colleagues (Mol Cancer. 2011 Jan 13;10:8) provides strong evidence that CCN1 (cyr61) is a potential therapeutic target in pancreatic cancer.

Homeostasis of the extracellular matrix (ECM) of tissues is regulated by controlling deposition and degradation of ECM proteins. The breakdown of ECM is essential in blastocyst implantation and embryonic development, tissue morphogenesis, menstrual shedding, bone formation, tissue resorption after delivery, and tumor growth and invasion. TGF-beta family members are one of the classes of proteins that actively participate in the homeostasis of ECM. Here, we report on the effect of lefty, a novel member of the TGF-beta family, on the homeostasis of extracellular matrix in a fibrosarcoma model. Fibroblastic cells forced to express lefty by retroviral transduction lost their ability to deposit collagen in vivo. This event was associated with down-regulation of the steady-state level of connective tissue growth factor that induces collagen type I mRNA. In addition, lefty transduction significantly decreased collagen type I mRNA expression and simultaneously increased collagenolytic, gelatinolytic, elastolytic, and caseinolytic activities in vivo by the transduced fibroblasts. These findings provide a new insight on the actions of lefty and suggest that this cytokine plays an active role in remodeling of the extracellular matrix in vivo.

In normal adult fibroblasts, transforming growth factor-beta (TGFbeta) induces the expression of connective tissue growth factor (CTGF). CTGF independently promotes fibroblast proliferation and matrix deposition, and in acute models of fibrosis promotes cell proliferation and collagen deposition acting synergistically with TGFbeta. In contrast to normal fibroblasts, fibroblasts cultured from fibrotic tissues express high basal levels of CTGF, even in the absence of added TGFbeta. Induction of transcription by TGFbeta requires the action of SMAD proteins. In this report we have investigated the role of SMADs in the TGFbeta-induction of CTGF in normal fibroblasts and in the elevated levels of CTGF expression found in dermal fibroblasts cultured from lesional areas of patients with scleroderma, a progressive fibrotic disorder that can affect all organs of the body. We have identified a functional SMAD binding site in the CTGF promoter. TGFbeta-induction of CTGF is dependent on SMAD3 and SMAD4 but not SMAD2 and is p300-independent. However, mutation of the SMAD binding site does not reduce the high level of CTGF promoter activity observed in dermal fibroblasts cultured from lesional areas of scleroderma patients. Conversely, the previously termed TGFbetaRE in the CTGF promoter is required for basal CTGF promoter activity in normal fibroblasts and for the elevated level of CTGF promoter activity in scleroderma fibroblasts. Thus, the maintenance of the fibrotic phenotype in scleroderma fibroblasts, as visualized by excess CTGF expression, appears to be independent of SMAD-dependent TGFbeta signaling. Furthermore, given CTGF's activities, the high level of CTGF expression observed in scleroderma lesions may contribute to the excessive scarring observed in this disorder.

Osteoarthritis (OA), or degenerative arthritis, is characterized by mechanical stress-induced changes in cartilage and bone. OA is a leading cause of chronic disability in North America and Europe. A recent study written by Blom and colleagues (Arthritis and Rheumatism 2009; 60:501–12) showed that elevated wnt signaling was observed in experimental OA as well as in patient samples. The authors found that the known wnt target WISP-1 (CCN4) was also overexpressed; CCN4 was sufficient to recapitulate an OA phenotype in vitro and in vivo, suggesting that CCN4 may be a novel target for drug intervention in OA. This commentary summarizes these exciting findings.

There is no treatment for fibrotic diseases, including the autoimmune disease systemic sclerosis (sclerderma, SSc). Although broad spectrum immune suppressants have little to no effect on the fibrosis in SSc, agents targeting specific proinflammatory agents are currently being considered as possible therapeutic approaches to combating SSc. B cells are lymphocytes that proliferate and secrete antibody molecules which drive the autoimmune response. CD20 is a B cell marker; the agent rituximab is an antibody against CD20. In a recent report by Bosello and colleagues (Arthritis Res. Ther. 12(2): R54, 2010), rituximab was tolerated in SSc patients and appeared to result in an improvement of the skin score and of clinical symptoms of SSc. This report is one of a series of recent studies suggesting that rituximab may be a possible treatment for SSc. This commentary summarizes these observations.

There is no treatment for fibrotic disease. TGFβ is known to promote fibrogenesis in vivo and in vitro, however, development of anti-fibrotic strategies targeting the TGFβ axis is problematic owing to the pleitropic nature of TGFβ action. Two recent papers (Kavian et al. 2010; Nyhan et al. 2010) suggest that the jagged/Notch pathway may selectively mediate fibrogenic properties of TGFβ and thus may represent a novel therapeutic approach to fibrosis for scleroderma and kidney fibrosis; these papers are the subject of this commentary.

The role of CCN proteins in vivo is only just becoming understood. A prototypical member of the CCN family, CCN3 suppresses proliferation. In a study in press, Shimoyama and colleagues show that mice lacking CCN3 have a hyperproliferative response to vascular injury. These data, along with other recent observations, suggest that CCN3 may represent a novel therapy for hyperproliferative diseases.

Fibrotic disease is a significant cause of mortality. CCN2 (connective tissue growth factor [CTGF]), a member of the CCN family of matricellular proteins, plays a significant role in driving the fibrogenic effects of cytokines such as transforming growth factor beta (TGFbeta). It has been proposed that other members of the CCN family can either promote or antagonize the action of CCN2, depending on the context. A recent elegant study published by Bruce Riser and colleagues (Am J Pathol. 174:1725-34, 2009) illustrates that CCN3 (nov) antagonizes the fibrogenic effects of CCN2. This paper, the subject of this commentary, raises the intriguing possibility that CCN3 may be used as a novel anti-fibrotic therapy.

The CCN family of matricellular signaling regulators shares a common domain structure. Variants of individual CCN proteins exist, which contain different combinations of these domains. Although mRNA splicing is likely to play a key role on CCN biology, this hypothesis has not been thoroughly tested. In a recent report, Hirschfeld and colleagues (Cancer Res 69:2082-90, 2009), show that CCN1 (cyr61) mRNA is normally present in a form in which intron 3 is retained. In cancers, or upon hypoxia, intron 3 is removed resulting in the appearance of CCN1 protein. The significance of this paper is discussed.

The matricellular protein CCN2 (connective tissue growth factor, CTGF) has been previously implicated in tumorigenesis. In pancreatic cancer cells, CCN2 expression occurs downstream of ras/MEK/ERK. Direct evidence that CCN2 mediates tumor progression in pancreatic cancer has been lacking. An exciting recent report by Bennewith et al. (Cancer Res 69:775-784, 2009) has used shRNA knockdown of CCN2 to illustrate that CCN2 contributes to growth of pancreatic tumor cells, both in vitro and in vivo. This report briefly summarizes these findings.

CCN2 (connective tissue growth factor [CTGF]/hypertrophic chondrocyte-specific gene product 24 [Hcs24]) is regulated at the transcriptional and posttranscriptional level. For example, an element in the its 3' untranslated region (3'-UTR) of the CCN2 mRNA controls message stability in chondrocytes. In a recent study, Mukudai et al. (Mol Cell Biol 28:6134-6147, 2008) purified and identified a trans-factor protein binding to the minimal repressive cis element in the 3'-UTR of ccn2 mRNA and identify this protein as the multifunctional nucleolar phosphoprotein nucleophosmin (NPM) This commentary summarizes these observations.

The CCN family of matricellular proteins directly or indirectly affects development and differentiation. A recent report written by Tan and colleagues (Am J Physiol Cell Physiol 295: C740–C751 2008) shows that CCN2 inhibits adipocyte differentiation. This commentary summarizes these observations.

A recent study by Gressner and colleagues suggested caffeine may block CCN2 expression in hepatic cells. This commentary briefly summarizes these observations.

There are several forms of hereditary human hair loss, known collectively as alopecias, the molecular bases of which are entirely unknown. A kindred with a rare, recessively inherited type of alopecia universalis was used to search for a locus by homozygosity mapping, and linkage was established in a 6-centimorgan interval on chromosome 8p12 (the logarithm of the odds favoring linkage score was 6.19). The human homolog of a murine gene,hairless, was localized in this interval by radiation hybrid mapping, and a missense mutation was found in affected individuals. Humanhairlessencodes a putative single zinc finger transcription factor protein with restricted expression in the brain and skin.

Background Scleroderma fibroblasts are characterized by elevated adhesive signaling. PTEN, a phosphatase that suppresses adhesive signaling, expression is reduced in scleroderma dermal fibroblasts. Previously, we showed that loss of PTEN expression in dermal mouse fibroblasts results in skin fibrosis associated with an elevated expression of the fibrotic marker CTGF/CCN2. . Objectives Our aim is to whether (a) loss of PTEN expression in fibroblasts also results in lung fibrosis ands (b) whether CCN2 mediates lung fibrosis caused by loss of PTEN. Methods A cre/lox system is used to delete PTEN and/or CCN2 in fibroblasts. Mice expressing a tamoxifen-dependent cre under the control of a fibroblast specific (COL1A2) promoter were generated that were homozygous for a loxP-PTEN allele and/or a loxP-CCN2 allele. Mice were injected with corn oil (to generate control mice) or tamoxifen (to delete PTEN and/or CCN2). Forty five days post-cessation of tamoxifen injection, lungs were harvested and subjected to histological and biochemical analyses to assess the effect of loss of PTEN and/or CCN2 on lung fibrogenesis (ie, Hematoxylin and eosin and trichrome stains, hydroxyproline/proline analysis, real time polymerase chain analysis, and immunohistological analyses with anti-type I collagen, anti-p-Akt, anti-CCN2, anti-phospho-Smad3 and anti-a-smooth muscle actin antibodies). Blood vessels were detected using anti-CD31 antibodies. Anti-NG2 antibodies were used to detect expansion of microvascular pericytes. Results Loss of PTEN expression resulted in lung fibrosis around the blood vessels. Loss of PTEN resulted in collagen and a-smooth muscle actin expression and deposition as visualized by immunohistochemical analysis (N=6, p Conclusions Loss of PTEN in fibroblasts results in lung fibrosis, in a fashion dependent on CCN2. Thus mice deficient in PTEN may represent a novel model for certain features of scleroderma. These results moreover indicate that elevated adhesive signaling (due to loss of PTEN expression) is sufficient for fibrogenesis and that CCN2 may represent a common downstream mediator of fibrosis, possibly due to its ability to promote expansion of a pericyte or pericyte-like cell population. Disclosure of Interest None Declared

PURPOSE. The precise role of a normal keratocyte in maintaining corneal structural integrity is unclear; it is generally considered to remain quiescent at the end of cell division. Given that integrins are essential for cell/extracellular matrix interactions, the authors tested the hypothesis that integrin expression by keratocytes is essential for corneal structure and function. METHODS. Using a tamoxifen-dependent cre recombinase expressed under the control of a fibroblast-specific promoter/ enhancer, the authors conditionally deleted the integrin 1 (Itgb1) gene in mouse keratocytes during the postnatal matrix maturation phase of the cornea. The effects of this deletion were monitored histologically and by macroscopic observation of the cornea. RESULTS. The resultant cornea shows an initial thinning of the stroma, reduced space between collagen fibrils, loss of epithelial layers and subsequent edema, thickening of Descemet’s membrane, and degenerative changes in the endothelial cell layer, with eventual scarring. These pathologic changes have some similarities to human corneal disease keratoconus. The phenotype did not develop when Itgb1 was deleted after complete corneal maturation. CONCLUSIONS. Loss of integrin 1 expression in keratocytes during the phase of stromal maturation results in corneal thinning and edema. Keratocyte-ECM interaction is essential for matrix maturation and thus in the maintenance of corneal structural integrity. This model has relevance in understanding corneal diseases such as keratoconus. (Invest Ophthalmol Vis Sci. 2011;52:7799‐7806) DOI:10.1167/iovs.10-6945

Background The mechanism underlying the ability of fibroblasts to contract a collagen gel matrix is largely unknown. Fibroblasts from scarred (lesional) areas of patients with the fibrotic disease scleroderma show enhanced ability to contract collagen relative to healthy fibroblasts. Thrombospondin 1 (TSP1), an activator of latent transforming growth factor (TGF)β, is overexpressed by scleroderma fibroblasts. In this report we investigate whether activation of latent TGFβ by TSP1 plays a key role in matrix contraction by normal and scleroderma fibroblasts. Methods We use the fibroblast populated collagen lattices (FPCL) model of matrix contraction to show that interfering with TSP1/TGFβ binding and knockdown of TSP1 expression suppressed the contractile ability of normal and scleroderma fibroblasts basally and in response to TGFβ. Previously, we have shown that ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mediates matrix contraction basally and in response to TGFβ. Results During mechanical stimulation in the FPCL system, using a multistation tensioning-culture force monitor (mst-CFM), TSP1 expression and p-ERK activation in fibroblasts are enhanced. Inhibiting TSP1 activity reduced the elevated activation of MEK/ERK and expression of key fibrogenic proteins. TSP1 also blocked platelet-derived growth factor (PDGF)-induced contractile activity and MEK/ERK activation. Conclusions TSP1 is a key mediator of matrix contraction of normal and systemic sclerosis fibroblasts, via MEK/ERK.

Following publication of our recent article [1], an error in Figure 5d was noticed. In Figure 5d, the β-actin blot corres ponding to α-SMA blot was captured on the chemi luminescence imaging system in an inverted orientation. Since this imager does not capture the protein marker, an error in the orientation of the membrane while capturing the picture of the blot occurred. During preparation of the fi gures for Figure 5d, the α-SMA blot was in right orientation but since orientation of β-actin blot was inverse as it was captured inversely resulted in non-corresponding β-actin blot with respect to α-SMA blot. We have rectifi ed this error now and β-actin blot now corresponds to α-SMA blot. Th e corrected fi gure 5 is given here as Figure 1 and a supplementary fi gure 1 (Additional fi le 1) showing another set of blots representing Figure 5d is also provided.

INTRODUCTION: Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H2 to PGE2. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrogenesis is largely unknown. Herein, we examine the role of mPGES-1 in a mouse model of skin scleroderma using mice deficient in mPGES-1. METHODS: Wild type (WT) and mPGES-1 null mice were subjected to the bleomycin model of cutaneous skin scleroderma. mPGES-1 expressions in scleroderma fibroblasts and in fibroblasts derived from bleomycin-exposed mice were assessed by Western blot analysis. Degree of fibrosis, dermal thickness, inflammation, collagen content and the number of α-smooth muscle actin (α-SMA)-positive cells were determined by histological analyses. The quantity of the collagen-specific amino acid hydroxyproline was also measured. RESULTS: Compared to normal skin fibroblasts, mPGES-1 protein expression was elevated in systemic sclerosis (SSc) fibroblasts and in bleomycin-exposed mice. Compared to WT mice, mPGES-1-null mice were resistant to bleomycin-induced inflammation, cutaneous thickening, collagen production and myofibroblast formation. CONCLUSIONS: mPGES-1 expression is required for bleomycin-induced skin fibrogenesis. Inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control the onset of fibrogenesis.

Deployment of the bicoid morphogen gradient in early Drosophila embryos requires the prelocalization of bicoid mRNA to the anterior pole of the egg. This anterior localization is mediated by a cis-acting localization signal contained within the 3 ′ untranslated region of the bicoid mRNA. Here we use a series of bicoid transgenes carrying small deletions in the 3 ′ untranslated region to survey for functional elements that constitute the localization signal. We identify and characterize one essential element, BLE1, which specifically directs the early steps of localization. In addition, we find that many deletions within the bicoid mRNA 3 ′ untranslated region impair but do not prevent localization. One such deletion specifically interferes with a later step in localization. Thus the bicoid mRNA localization signal appears to consist of multiple different elements, each responsible for different steps in the localization process.

It has now been 3 years since the inaugural issue of the Journal of Cell Communication and Signaling as published by Springer. Taking into consideration its predecessor, Cell Communication and Signaling (JCCS) published by Biomed Central, it has now been 7 years since the International CCN Society has sponsored a journal. The original intent of the International CCN Society was to create a journal disseminating knowledge concerning the CCN family of matricellular proteins. In 2003, it was perceived that the CCN family of proteins was under-recognized within the broader scientific community and was in need of a journal emphasizing their role. It is our belief that, in this regard, the journal has been a great success and has aided in the overall growth of interest in the CCN family as critical modifiers of signaling pathways involved with a wide range of physiological activities. In particular, we feel that the CCN family is now recognized by the translational research community as playing an essential role in human diseases including cancer and fibrosis by modifying cellular responses to signaling molecules including cytokines and extracellular matrix. Indeed, we feel that detailed knowledge of these interactions will be essential, in the future, for developing novel therapeutic approaches to combat disease. In 2010, we recognize that there is a need for a journal that aims to publish quality manuscripts focusing on the complex, context-dependent interactions between cells and their environment and the role of these interactions in disease. We have decided to alter and broaden the focus of JCCS to fill this gap. Thus, with this issue of JCCS, we are instituting several changes. First, in consultation with Springer, we have modernized the cover of JCCS, which will now change each quarter to include a photograph or illustration of a key concept published in the issue. Second, the membership of the editorial board has been altered to reflect the broader focus of the journal on translational research. Finally, to emphasize the continuity with the original scope of JCCS, Prof Bernard Perbal will remain as Executive Editor, but Prof Andrew Leask will now assume a new position as Scientific Editor. We hope that you will consider sending articles to JCCS. We continue to promise a rapid and fair review process focused on the quality of the science presented. Sincerely Andrew Leask Bernard Perbal

Fibrosis affects organs such as the skin, liver, kidney and lung and is a cause of significant morbidity. There is no therapy for fibrosis. Recent significant molecular insights into the signaling underlying fibrosis have been made. Transforming growth factor beta (TGF beta) signaling is a major contributor to fibrogenesis. The signaling mechanisms through which TGF beta induces fibrogenic responses have been under intense scrutiny. Moreover, the potent pro-fibrotic proteins endothelin-1 (ET-1) and CCN2 (connective tissue growth factor, CTGF) are believed to play an essential role in this process as downstream regulators or co-factors of TGF beta signaling. This review summarizes these recent crucial observations with emphasis on the disease scleroderma.

Vitamin A and other retinoids profoundly inhibit morphological and biochemical features of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, we examined retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. Our studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5' upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors alpha and gamma, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes.

The keratinocyte is a major cell type of the body, and in epidermis, keratinocytes have potential for future gene targeting and drug therapy. Despite the importance of keratinocytes in cell biology and medicine, little is known about the molecular mechanisms underlying keratinocyte-specific gene expression. Here, we report the first detailed characterization of the sequences and factors controlling expression of a human gene expressed specifically in keratinocytes. Using 5' upstream sequence of the human K14 keratin gene coupled to one of two reporter genes, we examined sequences necessary and sufficient for expression of K14 in both cultured human keratinocytes and in mitotically active basal keratinocytes of transgenic mouse epidermis. We demonstrated the existence of distal and proximal elements located 5' from the transcription initiation site of the hK14 gene, which when combined with a TATA box element, appear to act in concert to drive keratinocyte-specific expression. We examined the proximal region in detail. After using CAT assays to narrow a transcriptional activation element to within 110 bp, we demonstrated the existence of a keratinocyte nuclear factor which binds to a 10-bp palindrome, 5'-GCCTGCAGGC-3', within this domain. Using methylation interference analysis, we identified the G residues important for factor binding, and showed that point mutations in these G residues not only blocked factor binding but also resulted in decreased transcriptional activity of an hK14-CAT gene. The factor was most abundant in keratinocytes, was expressed at lower levels in some simple epithelial cell lines, and was not detected in fibroblasts or lymphoma cells. Moreover, the 10-bp sequence was similar to sequences found in the 5' upstream sequences of several other genes expressed in keratinocytes, and at least one of these genes, the human K1 gene, contained a sequence that competed with the hK14 proximal element for binding factor. Collectively, our data suggest that both the sequence and the nuclear factor that we have identified may be involved in controlling keratinocyte-specific expression in vitro and in vivo.

CCN2 is encoded by an immediate-early gene induced in mesenchymal cells during the formation of blood vessels, bone and connective tissue. It plays key roles in cell adhesion and migration, as well as matrix remodeling. CCN2 is overexpressed in fibrosis, arthritis and cancer; thus, an understanding of how to control CCN2 expression is likely to have importance in developing therapies to combat these pathologies. Previously, we found that the promoter sequence GAGGAATG is important for Ccn2 gene regulation in NIH 3T3 fibroblasts. In this report, we show that this sequence mediates activation of the CCN2 promoter by the ETS family of transcription factors. Endogenous Ets-1 binds this element of the CCN2 promoter, and dominant negative Ets-1 and specific Ets-1 small interfering RNA block induction of CCN2 expression by TGFβ. In the absence of added TGFβ1, Ets-1, but not the related fli-1, synergizes with Smad 3 to activate the CCN2 promoter. Whereas the ability of transfected Ets-1 to activate the CCN2 promoter is dependent on protein kinase C (PKC), Ets-1 in the presence of co-transfected Smad3 does not require PKC, suggesting that the presence of Smad3 bypasses the requirement of Ets-1 for PKC to activate target promoter activity. Our results are consistent with the notion that Smad3 and Ets-1 cooperate in the induction of the CCN2 promoter by TGFβ1. Antagonizing Ets-1 might be of benefit in attenuating CCN2 expression in fibrosis, arthritis and cancer, and may be useful in modulating the outcome of these disorders.

The autoimmune disease scleroderma (systemic sclerosis (SSc)) is characterized by extensive tissue fibrosis, causing significant morbidity. There is no therapy for the fibrosis observed in SSc; indeed, the underlying cause of the scarring observed in this disease is unknown. Transforming growth factor-β (TGFβ) has long been hypothesized to be a major contributor to pathological fibrotic diseases, including SSc. Recently, the signaling pathways through which TGFβ activates a fibrotic program have been elucidated and, as a consequence, several possible points for anti-fibrotic drug intervention in SSc have emerged.

The origin of fibrotic cells within connective tissue is unclear. For example, the extent to which microvascular pericytes contribute to the number of myofibroblasts present in dermal fibrosis in uncertain. Connective tissue growth factor (CTGF/CCN2) is a marker and mediator of fibrosis. In this report, we use an antibody recognizing CCN2 to assess the cell types in mouse dermis which express CCN2 in the bleomycin model of skin scleroderma. Control (PBS injected) and fibrotic (bleomycin-injected) dermis was examined for CCN2, alpha-smooth muscle actin (alpha-SMA) (to detect myofibroblasts), and NG2 (to detect pericytes) expression. Consistent with previously published data, CCN2 expression was largely absent in the dermis of control mice. However, upon exposure to bleomycin, CCN2 was observed in the dermis. Cells that expressed CCN2 were alpha-SMA-expressing myofibroblasts. Approximately 85% of myofibroblasts were NG2-positive, CCN2-expressing pericytes, indicating that pericytes significantly contributed to the presence of myofibroblasts in sclerotic dermis. Thus CCN2 is induced in fibrotic skin, correlating with the induction of myofibroblast induction. Moreover, CCN2-expressing pericytes significantly contribute to the appearance of myofibroblasts in bleomycin-induced skin scleroderma.

Connective tissue growth factor (CTGF, CCN2) is overexpressed in pancreatic cancer. We mapped the minimal CCN2 promoter active in PANC-1 cells, a human pancreatic cancer cell line. Within this region, Sp1, BCE-1 and Ets elements were important for the activity of the CCN2 promoter. Constitutive hyperactivated ras is a hallmark of cancers, including that of the pancreas. Treatment of PANC-1 cells with the MEK inhibitor U0126 or the Sp1 inhibitor mithramycin reduced CCN2 mRNA and promoter activity. Mutation of the BCE-1, but not Sp1 or Ets, site abolished the responsiveness of the CCN2 promoter to U0126. Overexpressing constitutively active MEK1 or ras activated CCN2 promoter activity. Thus CCN2 is likely to act downstream of ras in PANC-1 cells. CCN2 is overexpressed in cancer cells. Activated ras/MEK/ERK is a hallmark of cancer, and we have shown that the elevated CCN2 expression in pancreatic cancer cells is dependent on this pathway.

Connective tissue growth factor (CCN2), a member of the CCN family of proteins, is a cysteine-rich matricellular protein. Connective tissue growth factor is not normally expressed in dermal fibroblasts unless induced. The most potent inducer of connective tissue growth factor thus far identified is transforming growth factor beta. Connective tissue growth factor, however, is constitutively overexpressed by fibroblasts present in skin fibrotic lesions, including scleroderma. The overexpression of connective tissue growth factor present in fibrotic lesions contributes to the phenotype of scleroderma in that connective tissue growth factor promotes matrix deposition, and fibroblast adhesion and proliferation. In animal models, whereas either transforming growth factor beta or connective tissue growth factor alone produce only a transient fibrotic response, connective tissue growth factor and transforming growth factor beta act together to promote sustained fibrosis. Thus the constitutive overexpression of connective tissue growth factor by fibroblasts present in fibrotic lesions would be expected to contribute directly to chronic, persistent fibrosis. This review discusses recent information regarding insights into connective tissue growth factor biology and, using scleroderma as a model system, the part connective tissue growth factor might play in fibrotic disease.

Fibrosis represents a common pathway leading to organ failure and death in many diseases and has no effective therapy. Dysregulated repair and excessive tissue scarring provides a unifying mechanism for pathological fibrosis. The protein phosphatase and tensin homolog (PTEN) acts to dephosphorylate proteins, which promotes tissue repair and thus could be a key fibrogenic mediator. To test this hypothesis, we first showed that PTEN expression was reduced in skin fibroblasts from patients with the fibrotic autoimmune disease diffuse systemic sclerosis (dSSc). To evaluate whether this deficiency could be sufficient for fibrogenesis in vivo, we deleted PTEN in adult mouse fibroblasts. Compared with littermate control mice, loss of PTEN resulted in a 3-fold increase in dermal thickness due to excess deposition of collagen. PTEN-deleted fibroblasts showed elevated Akt phosphorylation and increased expression of connective tissue growth factor (CTGF/CCN2). Selective inhibition of the phosphatidylinositol 3-kinase/Akt pathway reduced the overexpression of collagen and CCN2 by PTEN-deficient fibroblasts. Overexpression of PTEN reduced the overexpression of type I collagen and CCN2 by dSSc fibroblasts. Thus, PTEN appears to be a potential in vivo master regulator of fibrogenesis; PTEN agonists may represent anti-fibrotic treatments.

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFbeta and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.

Background Transforming growth factor beta (TGFβ), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Even though the effects of TGFβ on the gene expression of several proteins have been investigated in several lung fibroblast cell lines, the global pattern of response to this cytokine in adult lung fibroblasts is still unknown. Methods We used Affymetrix oligonucleotide microarrays U95v2, containing approximately 12,000 human genes, to study the transcriptional profile in response to a four hour treatment with TGFβ in control lung fibroblasts and in fibroblasts from patients with idiopathic and scleroderma-associated pulmonary fibrosis. A combination of the Affymetrix change algorithm (Microarray Suite 5) and of analysis of variance models was used to identify TGFβ-regulated genes. Additional criteria were an average up- or down- regulation of at least two fold. Results Exposure of fibroblasts to TGFβ had a profound impact on gene expression, resulting in regulation of 129 transcripts. We focused on genes not previously found to be regulated by TGFβ in lung fibroblasts or other cell types, including nuclear co-repressor 2, SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), bone morphogenetic protein 4, and angiotensin II receptor type 1 (AGTR1), and confirmed the microarray results by real time-PCR. Western Blotting confirmed induction at the protein level of AGTR1, the most highly induced gene in both control and fibrotic lung fibroblasts among genes encoding for signal transduction molecules. Upregulation of AGTR1 occurred through the MKK1/MKK2 signalling pathway. Immunohistochemical staining showed AGTR1 expression by lung fibroblasts in fibroblastic foci within biopsies of idiopathic pulmonary fibrosis. Conclusions This study identifies several novel TGFβ targets in lung fibroblasts, and confirms with independent methods the induction of angiotensin II receptor type 1, underlining a potential role for angiotensin II receptor 1 antagonism in the treatment of lung fibrosis.

A major issue concerning clinical trials is the availability of standardized assays to evaluate drug efficacy. Ideally, such assays should test the effect of a putative drug on the expression of a biomarker in biological fluids. In a recent study by Kuiper et al. (PLOS One, 3(7): e2675). The relative levels of vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor [CTGF]) were examined in proliferative diabetic retinopathy (PDR). This paper is the subject of this commentary.