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PDF file - 126K, H&E staining of canine breast cancer tissues and western blot analysis of ADAM-12 in canine tissues

A disintegrin and metalloprotease domain-containing protein 12 (ADAM-12) is upregulated in many human cancers and promotes cancer metastasis. Increased urinary level of ADAM-12 in breast and bladder cancers correlates with disease progression. However, the mechanism of its induction in cancer remains less understood. Previously, we reported a Z-DNA–forming negative regulatory element (NRE) in ADAM-12 that functions as a transcriptional suppressor to maintain a low-level expression of ADAM-12 in most normal cells. We now report here that overexpression of ADAM-12 in triple-negative MDA-MB-231 breast cancer cells and breast cancer tumors is likely due to a marked loss of this Z-DNA–mediated transcriptional suppression function. We show that Z-DNA suppressor operates by interaction with methyl-CpG-binding protein, MeCP2, a prominent epigenetic regulator, and two members of the nuclear factor 1 family of transcription factors, NF1C and NF1X. While this tripartite interaction is highly prevalent in normal breast epithelial cells, both in vitro and in vivo, it is significantly lower in breast cancer cells. Western blot analysis has revealed significant differences in the levels of these 3 proteins between normal mammary epithelial and breast cancer cells. Furthermore, we show, by NRE mutation analysis, that interaction of these proteins with the NRE is necessary for effective suppressor function. Our findings unveil a new epigenetic regulatory process in which Z-DNA/MeCP2/NF1 interaction leads to transcriptional suppression, loss of which results in ADAM-12 overexpression in breast cancer cells. Cancer Res; 73(2); 736–44. ©2012 AACR.

PDF file - 91K, List of cancer-associated genes containing potential Z-DNA elements

Activation of mTOR is implicated in the development and progression of breast cancer. mTOR inhibition exhibited promising antitumor effects in breast cancer; however, its effect is compromised by several feedback mechanisms. One of such mechanisms is the upregulation of mTOR pathway in breast cancer cells. Despite the established role of mTOR activation in breast cancer, the status of total mTOR protein and its impact on the tumor behavior and response to treatment are poorly understood. Besides, the mechanisms underlying mTOR protein degradation in normal and cancer breast cells are still largely unknown. We and others found that total mTOR protein level is elevated in breast cancer cells compared to their nonmalignant counterparts. We have detected defective proteolysis of mTOR protein in breast cancer cells, which could, at least in part, explain the high level of mTOR protein in these cells. We show that metformin treatment in MCF‐7 breast cancer cells induced degradation of mTOR and sequestration of this protein in a perinuclear region. The decrease in mTOR protein level in these cells correlated positively with a concomitant inhibition of proliferation and migration potentials of these cells. These findings provided a novel mechanism for the metformin action in breast cancer treatment. Understanding the proteolytic mechanism responsible for mTOR level in breast cancer may pave the way for improving the efficacy of breast cancer treatment regimens and mitigating drug resistance as well as providing a basis for potential novel therapeutic modalities for breast cancer.

In the majority of breast cancers, overexpression and hyperactivation of Ras in the tumor microenvironment play significant role in promoting cancer cell growth, angiogenesis, and metastasis. We have previously shown that vascular endothelial growth factor (VEGF) expression in triple negative breast cancer cells is regulated, at least in part, by SAF-1 (serum amyloid A activating factor 1) transcription factor. In this study we show that transformation of normal MCF-10A breast epithelial cells by constitutively active, oncogenic Ras, induces the DNA-binding activity and transcription function of SAF-1. Furthermore, we show that inhibition of MEK/MAPK-signaling pathway prevents Ras-mediated activation of SAF-1. Interestingly, silencing of SAF-1 expression in breast cancer cells by SAF-1-specific short hairpin RNAs (shRNAs) significantly reduced H-Ras and K-Ras mRNA level. We show that SAF-1 is a direct transcriptional regulator of H-Ras and K-Ras and overexpression of SAF-1 increases H-Ras and K-Ras gene expression. Chromatin immunoprecipitation (ChIP) analyses demonstrated in vivo interaction of SAF-1 at highly purine-rich sequences present at the proximal promoter region, upstream of the transcription start site, in H-Ras and K-Ras genes. Previous studies have shown that these sequences are nuclease hypersensitive and capable of forming G4 quadruplex structure. Together, our results show the presence of a novel transactivating loop, in which, Ras and SAF-1 are interconnected. These findings will help defining molecular mechanisms of abnormal overexpression of Ras in breast tumors, which seldom show genetic Ras mutations.

Upregulation of ADAM-12, a novel member of the multifunctional ADAM family of proteins is linked to cancer, arthritis and cardiac hypertrophy. Basal expression of ADAM-12 is very low in adult tissues but rises markedly in response to certain physiological cues, such as during pregnancy in the placenta, during development in neonatal skeletal muscle and bone and in regenerating muscle. Studies on ADAM-12 regulation have identified a highly conserved negative regulatory element (NRE) at the 5′-UTR of human ADAM-12 gene, which acts as a transcriptional repressor. The NRE contains a stretch of dinucleotide-repeat sequence that is able to adopt a Z-DNA conformation both in vitro and in vivo and interacts with hZ α ADAR1 , a bona fide Z-DNA-binding protein. Substitution of the dinucleotide-repeat-element with a non-Z-DNA-forming sequence inhibited NRE function. We have detected a NRE DNA-binding protein activity in several tissues where ADAM-12 expression is low while no such activity was seen in the placenta where ADAM-12 expression is high. These observations suggest that interaction of these proteins with ADAM-12 NRE is critical for transcriptional repression of ADAM-12. We also show that the Z-DNA forming transcriptional repressor element, by interacting with these putative Z-DNA-binding proteins, is involved in the maintenance of constitutive low-level expression of human ADAM-12. Together these results provide a foundation for therapeutic down-regulation of ADAM-12 in cancer, arthritis and cardiac hypertrophy.

We reported earlier in a guinea pig model that exposure of 2-chloroethyl ethyl sulfide (CEES), a mustard gas analog, causes lung injury associated with the activation of tumor necrosis factor alpha (TNF-alpha), mitogen activated protein kinases (MAPK) signaling, and activator protein-1 (AP-1) transcription factor. Our earlier studies also revealed that antioxidant liposomes can be used as antidotes. Proinflammatory cytokines IL-1, IL-6, and TNF-alpha, either alone or in combination, can induce the activation of another group of transcription factors, namely SAF-1 (serum accelerator factor-1)/MAZ (Myc-associated zinc finger protein). Phosphorylation of SAF-1 via MAPK markedly increases its DNA-binding and transactivational potential. The objective of the present study was to investigate whether CEES exposure causes activation of IL-1 beta, IL-6, and SAF-1/MAZ and whether these effects can be prevented by antioxidant liposomes. A single dose (200 microL) of the antioxidant liposome mixture was administered intratracheally after 5 min of exposure of CEES (0.5 mg/kg). The animals were sacrificed either 1 h or 30 days after CEES exposure. CEES exposure caused an upregulation of proinflammatory cytokines IL-6 and IL-1 beta in the lung along with an increase in the activation of transcription factor SAF-1/MAZ. The antioxidant liposomes treatment significantly blocked the CEES-induced activation of IL-6, IL-1 beta, and SAF-1/MAZ. This might suggest that antioxidant liposomes might offer a potential therapeutic strategy against inflammatory diseases associated with activation of these bioactive molecules.

Previously we determined that inflammation responsive transcription factors AP-1 and SAF-1 synergistically regulate transcriptional induction of the MMP-1 gene. The present study investigated the underlying molecular mechanism of cooperativity between these two different groups of transcription factors. We present evidence that knockdown of SAF-1 by small interfering RNAs inhibits AP-1-mediated increase of human MMP-1 expression. The two key members of the AP-1 family of proteins, c-Fos and c-Jun, and SAF-1 form a ternary protein complex, which has markedly higher DNA binding activity than either a SAF-1 homodimer or a c-Fos/c-Jun heterodimer. The increased DNA binding activity of the ternary complex is translated into a striking enhancement of their transcriptional activity by which synergistic transcriptional induction of MMP-1 expression is achieved. The SAF-1.c-Fos.c-Jun ternary complex efficiently promotes transcription from both SAF-1 and AP-1 sites of human MMP-1 promoter. The physical interaction between SAF-1 and AP-1 was demonstrated both in vitro by Far-Western and antibody pulldown assays with recombinant proteins and in vivo by chromatin immunoprecipitation (ChIP), re-ChIP, and co-immunoprecipitation analyses. Two distinct but adjacent domains in SAF-1 are involved in protein-protein contact with c-Fos and c-Jun; one domain resides within two N-terminal polyalanine tracts, and the other is present within the first two zinc finger motifs. Together these findings delineate the mechanism of synergy and the essential role of SAF-1 and AP-1 in up-regulating human MMP-1 expression under various inflammatory conditions.

A number of risk factors including biomechanical stress on the articular cartilage imposed by joint overloading due to obesity, repetitive damage of the joint tissues by injury of the menisci and ligaments, and abnormal joint alignment play a significant role in the onset of osteoarthritis (OA). Genetic predisposition can also lead to the formation of defective cartilage matrix because of abnormal gene expression in the cartilage-specific cells. Another important biochemical event in OA is the consequence of inflammation. It has been shown that synovial inflammation triggers the synthesis of biological stimuli such as cytokines and growth factors which subsequently reach the chondrocyte cells of the articular cartilage activating inflammatory events in the chondrocytes leading to cartilage destruction. In addition to cartilage degradation, hypertrophy of the subchondral bone and osteophyte formation at the joint margins also takes place in OA. Both processes involve abnormal expression of a number of genes including matrix metalloproteinases (MMPs) for cartilage degradation and those associated with bone formation during osteophyte development. To address how diverse groups of genes are activated in OA chondrocyte, we have studied their induction mechanism. We present evidence for abundant expression of an inflammation-responsive transcription factor, SAF-1, in moderate to severely damaged OA cartilage tissues. In contrast, cells in normal cartilage matrix contain very low level of SAF-1 protein. SAF-1 is identified as a major regulator of increased synthesis of MMP-1 and -9 and pro-angiogenic factor, vascular endothelial growth factor (VEGF). While VEGF by stimulating angiogenesis plays a key role in new bone formation in osteophyte, increase of MMP-1 and -9 is instrumental for cartilage erosion in the pathogenesis of OA. Increased expression in degenerated cartilage matrix and in the osteophytes indicate for a key regulatory role of SAF-1 in directing catabolic matrix degrading and anabolic matrix regenerating activities.

The primary function of insulin is viewed as a hormone that controls blood glucose level. However, there is growing evidence that aberrant insulin level and insulin-mediated signaling can lead to cancer development and progression. The insulin-cancer relationship has stemmed from various observational and epidemiological studies, which linked higher incidence of cancer with central obesity, type II diabetes and other conditions associated with increased levels of circulating insulin, insulin resistance and hyperinsulinemic states. Increased risk of developing a range of cancers is also seen with a certain treatment options used to lower blood glucose level in diabetic patients. While metformin monotherapy has the lowest risk of developing cancer, in comparison, treatment with insulin or insulin secretagogues shows more likelihood to develop solid cancers. Cellular signaling initiated by insulin provides a clue regarding these diverse cellular outcomes. This review discusses how the insulin enacts such diverse physiological effects and the insulin-cancer relationship, with focus on the role of insulin signaling in cancer.

Abundantly expressed serum amyloid A (SAA) protein under chronic inflammatory conditions gives rise to insoluble aggregates of SAA derivatives in multiple organs resulting in reactive amyloid A (AA) amyloidosis, a consequence of rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis, familial Mediterranean fever, and Castleman’s disease. An inflammation-responsive transcription factor, SAF (for SAA activating factor), has been implicated in the sustained expression of amyloidogenic SAA under chronic inflammatory conditions. However, its role in the pathogenesis of AA amyloidosis has thus far remained obscure. In this paper we have shown that SAF-1, a major member of the SAF family, is abundantly present in human AA amyloidosis patients. To assess whether SAF-1 is directly linked to the pathogenesis of AA amyloidosis, we have developed a SAF-1 transgenic mouse model. SAF-1-overexpressing mice spontaneously developed AA amyloidosis at the age of 14 mo or older. Immunohistochemical analysis confirmed the nature of the amyloid deposits as an AA type derived from amyloidogenic SAA1. Furthermore, SAF-1 transgenic mice rapidly developed severe AA amyloidosis in response to azocasein injection, indicating increased susceptibility to inflammation. Also, during inflammation SAF-1 transgenic mice exhibited a prolonged acute phase response, leading to an extended period of SAA synthesis. Together, these results provide direct evidence that SAF-1 plays a key role in the development of AA amyloidosis, a consequence of chronic inflammation.

The placenta comprises a highly specialized trophoblast layer, which arises from the embryo and differentiates during embryonic development to perform specialized functions, e.g., synthesis of pregnancy-associated hormones, growth factors and cytokines. As there is no evidence of maternal acute-phase protein transplacental transfer and trophoblast plays an important role in regulating immune responses at the feto-maternal interface, the expression of acute-phase serum amyloid A (A-SAA) was investigated in human first trimester trophoblast and trophoblast-like JAR and Jeg-3 choriocarcinoma cells. We here show expression of cytokine receptors and cytokine-dependent induction of A-SAA in JAR and Jeg-3 cells. While interleukin-1alpha/beta is a major agonist for A-SAA expression in JAR, tumor necrosis factor-alpha is the predominant agonist in Jeg-3. First trimester trophoblast and JAR/Jeg-3 cells further express the human homolog of SAA-activating factor-1, a transcription factor involved in cytokine-mediated induction of A-SAA genes. A-SAA1 and A-SAA2 transcripts were increased in first trimester trophoblast during pregnancy weeks 10 and 12 suggesting that A-SAA plays a role during early fetal development.

Serum amyloid A-activating transcription factor-1 (SAF-1) plays a major role in regulating transcription of several inflammation-responsive genes, including SAA and matrix metalloproteinase-1, that are implicated in the pathogenesis of reactive secondary amyloidosis, atherosclerosis, and arthritis. SAF-1 is a 477-amino acid protein with six zinc fingers. Its activation during inflammatory condition by a phosphorylation event that leads to an altered structure suggested possible structural modification of this protein as a leading cause of higher activity. However, no information is available regarding structural features that might regulate its activity. Here, we have characterized its functional domains, delineating activation and repression modules, DNA binding, and nuclear localization activities. Using GAL4AD chimeras and a DNA-binding assay with proteins prepared from various deletion constructs, the core DNA-binding domain of SAF-1 is mapped between amino acids 282 and 361, which contain second, third, and fourth zinc fingers. Results from several deletion and point mutants using green fluorescent protein reporter show that SAF-1 contains two independent nuclear localization signals; one is composed of a stretch of basic amino acids, and the other is a bipartite signal located within the core DNA-binding domain. SAF-1 contains several negative and positively functioning transactivation modules clustered at the two ends of this protein. Removal of any one of the terminal negative modules renders the SAF-1 protein functionally very active. These findings suggest that the terminal repression modules act in conjunction to regulate the functional activity of this protein.

The transcription factor serum amyloid A-activating factor-1 (SAF-1) has been identified as a regulator of a number of cellular genes. To assess the pleiotropic role of SAF-1 in vivo, we generated SAF-1 transgenic mice, in which CMV immediate-early promoter was used to direct expression of the SAF-1 transgene in multiple organs. Our study shows that overexpression of SAF-1 predisposes animals to arthritis. Although SAF-1 transgenic mice do not spontaneously develop arthritis, they develop a severe form of arthritis when challenged with the Lyme disease agent Borrelia burgdorferi, which is known to promote arthritis development in both humans and mice. CMV-SAF-1 transgenic mice, upon B. burgdorferi infection, showed increased joint swelling and synovial inflammation compared with nontransgenic littermates. Immunohistochemical analysis of joint tissues collected 21 days after B. burgdorferi infection revealed colocalization of matrix metalloproteinase-1, a degradative enzyme that destroys type II collagen, a major architectural component of articular cartilage, and SAF-1 in both SAF-1 transgenic and nontransgenic mice. Further analysis by RNase protection assay and Western immunoblot demonstrated the presence of higher levels of matrix metalloproteinase-1 and SAF-1 in the inflamed joints of SAF-1 transgenic mice compared with their levels in nontransgenic mice. Consistent with these findings, reduced levels of proteoglycans were detected in the inflamed joint cartilage of transgenic mice, indicating damage to the cartilage structure. Together these results suggest a role of SAF-1 in the pathogenesis of inflammation-induced arthritis.

Inflammation-responsive transcription factor, serum amyloid A-activating factor 1 (SAF-1), has been shown to regulate several genes, including serum amyloid A, γ-fibrinogen, and matrix metalloproteinase 1, whose abnormal expression is associated with the pathogenesis of arthritis, atherosclerosis, and amyloidosis. Prolonged high level expression of SAF-1 in cultured cells failed to produce any stable cell line that overexpresses SAF-1. To test the fate of SAF-1-overexpressing cells, the cells were monitored for growth and morphological changes over time. The cells that were programmed to overproduce SAF-1 were found to undergo growth arrest and reduce DNA synthesis within 3 days after transfection. These cells undergo marked morphological changes from typical fibroblasts to round morphology and gradually cease to exist. Microarray analysis for cell cycle-specific genes in SAF1-transfected cells identified several candidate genes whose expression levels were altered during SAF-1 overexpression. Cdk inhibitor protein p21 was significantly affected by SAF-1; its expression level was highly induced by cellular conditions where SAF-1 is abundant. The increased level of p21 in the cell drives it to a growth arrest mode, a condition previously found to be controlled by p53. In this study we provide evidence that, similar to p53, SAF-1 is able to activate p21 gene expression by promoting transcription directly via its interaction with the p21 promoter. Together these data indicate that SAF-1 controls cell cycle progression via p21 induction, and pathophysiological conditions that favor overexpression of SAF-1, such as an acute inflammatory condition, can trigger cellular growth arrest.

Serum amyloid A-activating factor-1 (SAF-1), a Cys(2)His(2)-type zinc finger transcription factor, regulates inflammation-induced expression of serum amyloid A protein that is linked to the pathogenesis of reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. Here we report the identification of a novel splice variant, SAF-2, of the SAF family bearing strong sequence similarity to SAF-1. The N-terminal 426 amino acids of both SAF-1 and SAF-2 are identical containing two polyalanine tracts, one proline-rich domain, and six zinc fingers. However, the C terminus of SAF-2 containing two additional zinc fingers is different from SAF-1, which indicates the capability of different biochemical function. We show that SAF-2 interacts more avidly with the SAF-binding element, but its transactivation potential is much lower than SAF-1. Furthermore, co-expression of SAF-2 markedly suppresses SAF-1-regulated promoter function. Finally, we show that the level of SAF-2 protein is reduced during many inflammatory conditions, whereas the SAF-1 protein level remains unchanged. Together, these data suggest that the relative abundance of SAF-2 plays a critical role in the fine tuned regulation of inflammation-responsive genes that are controlled by SAF-1.

SAF-1, a zinc finger transcription factor, is activated by a number of inflammatory agents, including interleukin-1 (IL-1) and IL-6. It is involved in the cytokine-mediated transcriptional induction of serum amyloid A, an acute-phase plasma protein that is associated with the pathogenesis of reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. Here, we show that the mitogen-activated protein (MAP) kinase signaling pathway regulates cytokine-mediated induction of the DNA-binding activity and transactivation potential of SAF-1. Phosphorylation of endogenous SAF-1 in response to IL-1 and IL-6 was markedly inhibited by the addition of MAP kinase inhibitors. Consistent with this finding, we show that a consensus MAP kinase phosphorylation site, PPTP, within SAF-1 could be phosphorylated by MAP kinase in vitro. To analyze the contribution of MAP kinase in the activation of SAF-1, we prepared two independent mutant proteins in which the threonine residue of the PPTP motif was altered to either valine or alanine. These mutant proteins lost the ability to be phosphorylated by MAP kinase both in vivo and in vitro and exhibited a significantly reduced ability to promote expression of the SAF-1-regulated promoter. While the DNA-binding activity of wild-type SAF-1 protein was markedly increased upon phosphorylation with MAP kinase, no such increase could be detected with the mutant SAF-1 proteins. Further analysis with the GAL-4 reporter system showed that mutation of the MAP kinase phosphorylation site considerably lowers the transactivation potential of SAF-1. Together, these results show that activation of SAF-1 in response to IL-1 and -6 is mediated via MAP kinase-regulated phosphorylation.

The serum amyloid A activating factor (SAF) was identified as a family of inducible transcription factors that is activated by many mediators of inflammation. Its activation involves a phosphorylation event, whose mechanism is not fully understood. Here, we show that cAMP treatment of several cell types, including mouse liver-derived BNL CL.2, human monocyte-derived THP-1, and a primary culture of vascular smooth muscle cells from porcine aorta, activated cellular SAF's ability to bind DNA. The protein kinase A (PKA) activity in cytoplasmic extracts of cAMP-treated cells was responsible for the potentiation of the DNA-binding activity of the cellular SAF proteins. Furthermore, treatment of nuclear extracts of untreated cells with purified PKA increased the DNA-binding activity of cellular SAF proteins, and specific inhibitors of PKA abrogated the enhanced DNA-binding ability of SAF in the cAMP-treated cells. Consistent with these findings, overexpression of the catalytic subunit of PKA markedly increased expression of the SAF-regulated promoter. These results imply a functional role for the previously detected protein-protein interaction between SAF-1 transcription factor and the catalytic subunit of PKA and further demonstrate the consequences of cAMP-mediated signaling for the expression of SAF-regulated genes.

Metastatic spread of cancer is a leading cause for the loss of life from this disease. In metastatic cascade, cells undergo multifaceted phenotypic transformations that include breakdown of extracellular matrix (ECM) encasing the tumor, cancer cell migration and invasion of surrounding tissues, and relocation of cancer cells in secondary organs. Recently a multitasking protein, A Disintegrin and Metalloprotease-12 (ADAM-12), has attracted particular interest because of its potential roles in tumor growth and development by facilitating remodeling of extracellular matrix and cell migration that are so essential for cancer growth and metastasis. ADAM-12 is an active metalloproteinase; it regulates release of growth factors and is capable of promoting cell–cell and cell–matrix adhesion and cell signaling as well. Overexpression of ADAM-12 is reported in many types of human cancers. Furthermore, a statistical correlation between the urinary levels of ADAM-12 in breast and bladder cancer patients and cancer progression has been found. These results suggested that ADAM-12 could be used as a diagnostic marker. In addition to cancers, increase of ADAM-12 expression is linked to the pathogenesis of osteoarthritis, cardiac hypertrophy, and Alzheimer’s disease, as well as during high-fat diet-induced obesity. This review is meant to provide a broad overview of the regulatory pathways by which ADAM-12 could be expressed to contribute towards tumor development, accelerate tumor progression, and metastasis. A better understanding of the regulation of this multifunctional protein that could well be used as a new therapeutic option.

The transcription factor serum amyloid A (SAA)-activating factor (SAF), a family of zinc finger proteins, plays a significant role in the induced expression of the SAA gene. Activity of SAF is regulated by a phosphorylation event involving serine/threonine protein kinase (Ray, A., Schatten, H., and Ray, B. K. (1999) J. Biol. Chem. 274, 4300-4308; Ray, A., and Ray, B. K. (1998) Mol. Cell. Biol. 18, 7327-7335). However, the identity of the protein kinase has so far remained unknown. Induction of SAA by phorbol 12-myristate 13-acetate, a known agonist of protein kinase C (PKC), suggested a potential role of the PKC signaling pathway in the activation process. The DNA binding activity of endogenous SAF was increased by agonists of PKC. In vitro phosphorylation of SAF-1 by PKC-beta markedly increased its DNA binding ability. Consistent with these findings, treatment of cells with activators of PKC or overexpression of PKC-betaII in transfected cells increased expression of an SAF-regulated promoter. Further analysis with a GAL4 reporter system indicated that PKC-mediated phosphorylation mostly increases the DNA binding activity of SAF-1. Together these data indicated that the PKC signaling pathway plays a major role in controlling expression of SAF-regulated genes by increasing the interaction between promoter DNA and phosphorylated SAF.

The serum amyloid A (SAA) protein has been implicated in the pathogenesis of several chronic inflammatory diseases. Its induction mechanism in response to a chronic inflammatory condition was investigated in rabbits following multiple s.c. injections of AgNO3 over a period of 35 days. During unremitting exposure to inflammatory stimulus, a persistently higher than normal level of SAA2 expression was seen in multiple tissues. Induction of SAA was correlated with higher levels of several transcription factor activities. Increased SAA-activating factor (SAF) activity was detected in the liver, lung, and brain tissues under both acute and chronic inflammatory conditions. In the heart, kidney, and skeletal muscle tissues, this activity remained virtually constant. In contrast, CCAAT enhancer binding protein (C/EBP) DNA-binding activity was transiently induced in selective tissues. Higher than normal NF-κB DNA-binding activity was detected in the lung and to a lesser extent in the liver and kidney tissues under both acute and chronic conditions. This result suggested that C/EBP, SAF, and NF-κB are required for transient acute phase induction of SAA whereas SAF and NF-κB activities are necessary for persistent SAA expression during chronic inflammatory conditions.

The serum amyloid A (SAA) protein has been implicated in the progression and pathogenesis of rheumatoid arthritis through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are present at significantly high level in the synovial fluid of arthritis patients. These cytokines induced phenotypic changes in synovial cells, promoting protrusion and increased cellular contact. Induction of SAA under this condition is mediated by promoter elements located between -254 and -226, which contains binding sites for transcription factors Sp1 and SAA activating sequence binding factor (SAF). Mutation of these sequences abolishes SAA promoter response to IL-1 and IL-6. The role of Sp1 in SAA induction was demonstrated by increased DNA binding activity, phosphorylation, and increased protein content of Sp1 during cytokine treatment. Sp1 interacts with the SAA promoter in association with SAF as an SAF. Sp1 heteromeric complex. Furthermore, using a phosphatase inhibitor, we demonstrated increased transactivation potential of both Sp1 and SAF as a consequence of a phosphorylation event. These results provide first evidence for cytokine-mediated activation of Sp1 in synovial fibroblast cells and its participation in regulating SAA expression by acting in conjunction with SAF.

Minimally modified low-density lipoprotein (MM-LDL) is regarded as a major risk factor for the development of atherosclerosis. In this report, we show that this lipoprotein complex can induce expression of an inflammatory protein, serum amyloid A (SAA), in monocyte/macrophage cells, a key cell type implicated in the pathogenesis of atherosclerosis. By promoter function analysis and site-directed mutagenesis, we have located promoter regions responsive to MM-LDL action. Using electrophoretic mobility shift, antibody ablation/supershift, and Western blot assays, we showed that induction of SAA by MM-LDL is mediated via activation of SAS binding factor (SAF) and C/EBP transcription factors. We further show that tamoxifen, a downregulator of CD36, one of the major scavenger receptors which binds MM-LDL, can inhibit MM-LDL-mediated SAA induction in THP-1 cells. This finding suggests that CD36 participates in the manifestation of the inflammatory effects of MM-LDL. Our experiments provide the first evidence for transcription factor activation by MM-LDL.

Serum amyloid A (SAA), a plasma protein inducible in response to many inflammatory conditions, is associated with the pathogenesis of several diseases including reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. We have previously reported an element of the SAA promoter, designated SAA-activating sequence (SAS), that is involved in the inflammation-induced SAA expression, and a nuclear factor, SAS-binding factor (SAF), that interacts with the SAS element has been identified previously (A. Ray and B. K. Ray, Mol. Cell. Biol. 16:1584-1594, 1996). To evaluate how SAF is involved in SAA promoter activation, we have investigated structural features and functional characteristics of this transcription factor. Our studies indicate that SAF belongs to a family of transcription factors characterized by the presence of multiple zinc finger motifs of the Cys2-His2 type at the carboxyl end. Of the three cloned SAF cDNAs (SAF-1, SAF-5, and SAF-8), SAF-1 isoform showed a high degree of homology to MAZ/ZF87/Pur-1 protein while SAF-5 and SAF-8 isoforms are unique and are related to SAF-1/MAZ/ZF87/Pur-1 at the zinc finger domains but different elsewhere. Although structurally distinct, all members are capable of activating SAS element-mediated expression and display virtually identical sequence specificities. However, varying levels of expression of members of this gene family were observed in different tissues. Functional activity of SAF is regulated by a posttranslational event as SAF DNA-binding and transactivation abilities are increased by a protein phosphatase inhibitor, okadaic acid, and inhibited by a protein kinase inhibitor, H7. Consistent with this observation, increased DNA binding of the cloned SAF and its hyperphosphorylation, in response to okadaic acid treatment of the transfected cells, were observed. Taken together, our results suggest that, in addition to tissue-specific expression, SAFs, a family of zinc finger transcription factors, undergo a modification by a posttranslational event that confers their SAA promoter-binding activity and transactivation potential.

Serum amyloid A (SAA) is a plasma protein that is associated with many inflammatory diseases including amyloidosis, arthritis, and atherosclerosis. SAA level is significantly increased during inflammatory condition, and such abnormal expression of this protein is linked to the pathogenesis of the above-mentioned diseases. A promoter element, designated as SAA-activating sequence (SAS), located between −280 and −226 has been implicated in the induction mechanism and a nuclear factor, SAS-binding factor (SAF), has been shown to bind to this region. In this report, using a cloned SAF gene in transient transfection assay, we provide evidence that SAF potentiates SAA gene expression through SAS element. Furthermore, we show that during lipopolysaccharide-mediated induction of SAF, heteromeric complex with transcription factor Sp1 is formed. Transfection assays using both transcription factor genes have demonstrated that SAF-Sp1 heteromer is a highly potent transactivator of SAA expression.

Serum amyloid A (SAA) has been linked to atherosclerosis because of its ability to remodel high-density lipoprotein by the depletion of apolipoprotein A1, its ability to bind cholesterol, and its presence in the atherosclerotic plaques of coronary and carotid arteries. In the present study, we investigated the induction mechanism of SAA gene in THP-1 monocyte/macrophage cells which play a critical role in the development of atherosclerotic fatty streak and plaque formation. We and others have shown that SAA gene is induced in monocyte/macrophage cells by lipopolysaccharide (LPS). By promoter function analysis, we show that the SAA promoter sequence between -280 and -226 can confer LPS responsiveness. Gel electrophoretic mobility shift assay detected an induced DNA-binding activity in these cells in response to LPS. Characterization of the DNA-binding protein by UV cross-linking, Southwestern blot, and antibody ablation/supershift assays revealed that it is similar to a recently reported nuclear factor designated SAF. These results demonstrated that LPS-mediated SAA gene induction in monocyte/macrophage cells is primarily due to the induction of SAF activity.

Vascular endothelial growth factor (VEGF) is recognized as an important angiogenic factor that promotes angiogenesis in a series of pathological conditions, including cancer, inflammation, and ischemic disorders. We have recently shown that the inflammatory transcription factor SAF-1 is, at least in part, responsible for the marked increase of VEGF levels in breast cancer. Here, we show that SAF-1-mediated induction of VEGF is repressed by KLF-4 transcription factor. KLF-4 is abundantly present in normal breast epithelial cells, but its level is considerably reduced in breast cancer cells and clinical cancer tissues. In the human VEGF promoter, SAF-1- and KLF-4-binding elements are overlapping, whereas SAF-1 induces and KLF-4 suppresses VEGF expression. Ectopic overexpression of KLF-4 and RNAi-mediated inhibition of endogenous KLF-4 supported the role of KLF-4 as a transcriptional repressor of VEGF and an inhibitor of angiogenesis in breast cancer cells. We show that KLF-4 recruits histone deacetylases (HDACs) -2 and -3 at the VEGF promoter. Chronological ChIP assays demonstrated the occupancy of KLF-4, HDAC2, and HDAC3 in the VEGF promoter in normal MCF-10A cells but not in MDA-MB-231 cancer cells. Co-transfection of KLF-4 and HDAC expression plasmids in breast cancer cells results in synergistic repression of VEGF expression and inhibition of angiogenic potential of these carcinoma cells. Together these results identify a new mechanism of VEGF up-regulation in cancer that involves concomitant loss of KLF-4-HDAC-mediated transcriptional repression and active recruitment of SAF-1-mediated transcriptional activation. Background: VEGF plays an important role in tumor growth and metastasis. Results: KLF-4 suppresses VEGF expression in normal breast cells by recruiting HDACs. Cancer cells do not recruit HDACs due to low KLF-4 levels. Conclusion: Loss of KLF-4-HDAC-mediated suppression and active recruitment of SAF-1 causes cancer cell-specific VEGF expression. Significance: Findings identified a previously unknown mechanism of VEGF regulation and revealed new therapeutic targets.

Osteoarthritis (OA) is a disease that damages the joint tissues and often destroys the cartilage lining of the joint tissue irreversibly. This leads to loss of joint function making movement difficult and crippling the affected individuals. OA primarily affects elderly population and with increasing aging human population in the world it is going to affect a very large portion of the population in the near future. Unfortunately there is no current method of treatment of this disease except by suppressing the pain with pain-killer and occasional use of steroid to reduce excruciating pain. More radical treatment and often the only remaining option is surgery where affected joint tissue is removed and replaced with artificial prosthetics embedded into the bone, a procedure called arthroplasty. The surgical procedure is costly and sometimes associated with recurring post-operative problems. New approaches to control and cure OA could come from a better understanding of the cellular and subcellular events in the joint tissues of OA patients. Since cartilage depletion is a major pathological event in the progression of OA, much attention has been given to the cellular mechanisms responsible for the cartilage damage. This review will address this cellular process by focusing on enzymes which are activated in the cartilage tissue and cause its depletion.

Serum amyloid A (SAA) is a plasma protein which has been associated with several diseases, including amyloidosis, arthritis, and atherosclerosis, and its abnormal expression, particularly in nonhepatic cells, is implicated in the pathogenesis of these diseases. Transfection and DNA-binding studies were performed to investigate the mechanism controlling cytokine-induced, nonhepatic expression of the SAA gene. We have identified a novel promoter, located between positions -280 and 224, that confers interleukin-6 (IL-6) inducibility to an SAA-chloramphenicol acetyltransferase reporter gene in both nonhepatic and hepatic cells. DNase I protection assays revealed, within this region, three homologous highly pyrimidine rich octanucleotide sequence motifs, termed SAA-activating sequences (SAS). Specific mutations within these three SAS motifs severely reduced IL-6-mediated induction of the reporter gene in transfected nonhepatic cells but not in liver cells. A nuclear factor activated by IL-6 in both hepatic and nonhepatic cells efficiently interacts with the SAS. The induction kinetics and cycloheximide sensitivity of this SAS-binding factor (SAF) suggested that de novo synthesis of this factor itself or an activator protein is essential. Loss of DNA-binding ability as a result of in vitro dephosphorylation, induction of SAA-chloramphenicol acetyltransferase reporter gene activity in the presence of genistein, a protein kinase inhibitor, further indicate that a phosphorylation step is necessary for the activation of SAF. Our results suggest that SAF is a key regulator of cytokine-mediated SAA gene expression in some nonhepatic cells.

A disintegrin and metalloprotease domain-containing protein 12 (ADAM-12) is upregulated in many human cancers and promotes cancer metastasis. Increased urinary level of ADAM-12 in breast and bladder cancers correlates with disease progression. However, the mechanism of its induction in cancer remains less understood. Previously, we reported a Z-DNA–forming negative regulatory element (NRE) in ADAM-12 that functions as a transcriptional suppressor to maintain a low-level expression of ADAM-12 in most normal cells. We now report here that overexpression of ADAM-12 in triple-negative MDA-MB-231 breast cancer cells and breast cancer tumors is likely due to a marked loss of this Z-DNA–mediated transcriptional suppression function. We show that Z-DNA suppressor operates by interaction with methyl-CpG-binding protein, MeCP2, a prominent epigenetic regulator, and two members of the nuclear factor 1 family of transcription factors, NF1C and NF1X. While this tripartite interaction is highly prevalent in normal breast epithelial cells, both in vitro and in vivo, it is significantly lower in breast cancer cells. Western blot analysis has revealed significant differences in the levels of these 3 proteins between normal mammary epithelial and breast cancer cells. Furthermore, we show, by NRE mutation analysis, that interaction of these proteins with the NRE is necessary for effective suppressor function. Our findings unveil a new epigenetic regulatory process in which Z-DNA/MeCP2/NF1 interaction leads to transcriptional suppression, loss of which results in ADAM-12 overexpression in breast cancer cells. Cancer Res; 73(2); 736–44. ©2012 AACR.

alpha 1-Antitrypsin (alpha 1-AT) is one of the major proteinase inhibitors in serum. Its primary physiological function is to inhibit neutrophil elastase activity in lung, but it also inhibits other serine proteases including trypsin, chymotrypsin, thrombin, and cathepsin. We have previously reported a novel alpha 1-AT, S-2 isoform, from rabbit that is induced up to 100-fold in the liver during acute inflammatory condition (Ray, B. K., Gao, X., and Ray, A. (1994) J. Biol. Chem. 269, 22080-22086). Here, we present evidence that the expression of this alpha 1-AT S-2 gene is also induced in lipopolysaccharide (LPS)-treated peripheral blood monocytes. From the cloned genomic DNA, we have identified a distal LPS-responsive enhancer located between -2438 and -1990 base pairs upstream of the transcription start site. In vitro DNA-binding studies demonstrated an interaction of an LPS-inducible NF-kappa B-like nuclear factor with a kappa B-element present in this enhancer region. Antibodies against p65 and p50 subunits of NF-kappa B supershifted the DNA-protein complex. A mutation of the NF-kappa B-binding element virtually abolished the LPS-responsive induction of the chimeric promoter in monocytic cells. Furthermore, overexpression of NF-kappa B induced the wild-type promoter activity. Taken together, these results demonstrated that during LPS-mediated inflammation, NF-kappa B/Rel family of transcription factors play a crucial role in the transcriptional induction of the inflammation responsive alpha 1-AT gene.

Many studies have tested the consumption of foods and supplements to reduce exercise-induced muscle damage, but fasting itself is also worthy of investigation due to reports of beneficial effects of caloric restriction and/or intermittent fasting on inflammation and oxidative stress. This preliminary investigation compared indicators of exercise-induced muscle damage between upper-body untrained participants (N = 29, 22 yrs old (SD = 3.34), 12 women) who completed 8 hour water-only fasts or ate a controlled diet in the eight hours prior to five consecutive laboratory sessions. All sessions were conducted in the afternoon hours (i.e., post meridiem) and the women completed the first session while in the follicular phase of their menstrual cycles. Measures of muscle pain, resting elbow extension, upper arm girth, isometric strength, myoglobin (Mb), total nitric oxide (NO), interleukin 1beta (IL1b), and tumor necrosis factor alpha (TNFa) were collected before and after eccentric contractions of the non-dominant elbow flexors were completed. The fasting group’s loss of elbow extension was less than the post-prandial group (p < .05, eta2 = .10), but the groups did not change differently across time for any other outcome measures. However, significantly higher NO (p < .05, eta2 = .22) and lower TNFa (p < .001, eta2 = .53) were detected in the fasting group than the post-prandial group regardless of time. These results suggest intermittent fasting does not robustly inhibit the signs and symptoms of exercise-induced muscle damage, but such fasting may generally affect common indirect markers of muscle damage.

Tumor microenvironment (TME) plays a critical role in tumor growth, invasion and metastasis. In TME, epidermal growth factor receptor (EGFR) family members, including HER1, HER2, HER3 and HER4, are involved in determining aggressive growth of breast cancer due to their ability to transduce the growth promoting functions of growth factors. This activity is potentiated by the over-expression of these receptor molecules in cancer cells. To reduce the activity of EGFR molecules, various inhibitors have been developed. EGFR/HER1 tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show antitumor activity but these drugs have not meet their primary goal of improved survival in the overall patient population. A similar TKI, lapatinib, has shown some limited success in breast cancer. While these therapeutic options focus on reducing the functional activity of EGFR protein, the fundamental and major problem of over-expression of EGFR in cancer cells has not been addressed. In two-thirds of aggressive breast cancer patients, transcriptional induction of EGFR causes high EGFR/HER1 level. Therefore, the goal of this study is to identify a molecular mechanism by which EGFR is over-expressed in breast cancer cells. To understand how transcriptional induction might occur, we have explored a novel biosynthetic pathway for EGFR over-expression. Our findings reveal that EGFR promoter is significantly more active in MDA-MB-231 cells in comparison to MDA-MB-468 cells. When transcription factor SAF-1 was ectopically expressed in these cells, EGFR promoter activity was further increased in MDA-MB-231 cells. Since MDA-MB-231 cells contain a highly active form of Ras, the data suggested a possible Ras-mediated activation of SAF-1 which in turn induces EGFR expression. Consistent with these findings, inhibition of K- and H-Ras, by using both siRNA and CRISPR/Cas9-mediated knock-out systems, reduced the expression of EGFR in MDA-MB-231 cells. The effect was more profound when K-Ras was targeted for inhibition. Since SAF-1/MAZ is seen to be activated by Ras, our data implicates K-Ras - SAF-1/MAZ - EGFR axis in breast cancer cell growth. Our findings may provide new targets for breast cancer therapy. Supported by grants from College of Veterinary Medicine Faculty Research Award Grant and MU Center for Botanical Interaction Studies Pilot Project Grant. Citation Format: Alpana Ray, Brett Havis, Bimal Ray. A novel regulatory mechanism involving Ras-mediated activation of the zinc-finger transcription factor, SAF-1/MAZ induces EGFR/HER1 expression in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A65.

alpha 1-Antitrypsin is a major plasma proteinase inhibitor whose primary function is to control the proteolytic activity of neutrophil elastase that hydrolyzes structural proteins. This protein, in rabbit is expressed as three isoforms designated as F, S-1, and S-2. An inducible form of this protein has been cloned from an acute-phase cDNA library of rabbit liver. Structural study has revealed that the cloned cDNA is the S-2 isoform previously identified by partial peptide sequence analysis. The amino acid sequence of the reactive center of the S-2 form is not conserved and thus can be categorized as of unorthodox type. mRNA analysis has indicated that the transcription of the S-2 isoform which is normally present at a very low concentration increases more than 100-fold under inflammatory conditions while the expression of the F and S-1 isoforms changes about 1.5-fold under similar conditions. The S-2 isoform is biologically active and capable of inhibiting both elastase and chymotrypsin. The high level of induction of this active isoform of alpha 1-antitrypsin under inflammatory conditions and its apparent resistance to oxidation-mediated inactivation, common to the orthodox forms of alpha 1-antitrypsin, make the S-2 isoform suitable for gene therapy in diseases associated with alpha 1-antitrypsin deficiency.

Transcription of the gene encoding alpha 1-acid glycoprotein is highly induced during acute inflammation which has been previously shown to be mediated by some inducible members of the CCAAT-enhancer-binding (C/EBP) transcription-factor family. In this study, we demonstrate that the involved inducible C/EBP isoforms are C/EBP-beta and C/EBP-delta, and together they control the high-level induction of the alpha 1-acid glycoprotein gene in response to inflammatory signals. We observed that dephosphorylation severely inhibits the DNA-binding ability of C/EBP-delta and its transactivating potential increases in the presence of cellular phosphatase inhibitors, such as okadaic acid and sodium orthovanadate. These results suggest that C/EBP-delta is regulated by phosphorylation. Transient transfections using expression vectors of C/EBP-alpha, C/EBP-beta and C/EBP-delta have shown that while individually all three isoforms can transactivate the alpha 1-acid glycoprotein-chloramphenicol-acetyltransferase gene transcription, co-expression of C/EBP-alpha and C/EBP-beta isoforms results in lower levels of reporter gene expression than the levels predicted from their additive transactivation level. In vitro DNA-binding studies have shown that C/EBP-alpha and C/EBP-beta isoforms both interact and form complexes with the alpha 1-acid glycoprotein gene C/EBP-binding element under normal noninduced conditions during which alpha 1-acid glycoprotein is expressed at a very low level. Higher than additive levels of reporter gene expression are observed when combinations of C/EBP-delta and C/EBP-beta or C/EBP-delta and C/EBP-alpha are used. Together, these data demonstrate that C/EBP-beta and C/EBP-delta are the major proteins responsible for the acute-phase induction of alpha 1-acid-glycoprotein gene expression and they require phosphorylation for transactivation potential.

Serum amyloid A (SAA) is a plasma protein whose synthesis is markedly increased in the liver during the inflammatory process. Previous analysis of SAA promoter function implicated the involvement of the CCAAT/enhancer-binding protein (C/EBP) in controlling this process. In this study, using antibodies against three C/EBP isoforms in DNA-binding and Western blot (immunoblot) assays, we found that in response to inflammatory signals, both C/EBP-delta and C/EBP-beta are induced and that their interactions with the SAA promoter element are necessary for the increased SAA gene expression. Cotransfections of liver cells with an SAA promoter-linked reporter chloramphenicol acetyltransferase gene and murine sarcoma virus-expressed C/EBP-delta or C/EBP-beta confirm such phenomena. The increased transactivating ability in the presence of the cellular phosphatase inhibitors okadaic acid and sodium orthovanadate, coupled with the observation that dephosphorylation severely inhibits the DNA-binding ability in vitro, implicates a role of phosphorylation in the regulation of the activities of the C/EBP-delta isoform. Consistent with these findings, we have detected higher levels of DNA-binding activity of C/EBP-delta prepared from cells treated with phosphatase inhibitors. We also present evidence that C/EBP-delta is a phosphoprotein. These results suggest that C/EBP-delta is regulated by phosphorylation and, in conjunction with C/EBP-beta, is one of the major proteins responsible for the increased transcription of the SAA gene in response to inflammatory stimuli.

Angiogenesis plays a significant role in cancer by providing increased blood supply to the affected tissues and thus bringing in growth factors, cytokines, and various nutrients for tumor growth. VEGF is the most prominent angiogenic agent that is markedly induced in cancer. Induction of VEGF has been widely studied but as cancer cells are quite adept at acquiring new alternative processes to circumvent surrounding environmental pressures, our understanding of the molecular mechanisms regulating VEGF expression in cancer, especially in triple-negative breast cancer cells, remains incomplete. Here, we present evidence of a novel mode of VEGF induction in triple-negative MDA-MB-231 breast cancer cells that is regulated by serum amyloid A activating factor 1 (SAF-1) transcription factor. Inhibition of SAF-1 by antisense short hairpin RNA profoundly reduces VEGF expression along with reduction in endothelial cell proliferation and migration. By both in vitro and in vivo molecular studies, we show that the effect of SAF-1 is mediated through its direct interaction with the VEGF promoter. In correlation, DNA-binding activity of SAF-1 is found to be significantly higher in MDA-MB-231 breast cancer cells. Examination of several breast cancer samples further revealed that SAF-1 is overexpressed in clinical breast cancer tissues. Taken together, these findings reveal that SAF-1 is a hitherto unrecognized participant in inducing VEGF expression in triple-negative breast cancer cells, an aggressive form of breast cancer that currently lacks effective treatment options. Suppression of SAF-1 activity in these cells can inhibit VEGF expression, providing a possible new method to control angiogenesis. Mol Cancer Res; 9(8); 1030–41. ©2011 AACR.

Serum amyloid A (SAA) protein synthesis is highly induced in acute inflammatory conditions. Such induction process is primarily regulated at the transcriptional level and large amount of SAA mRNA has been found to accumulate in rabbit liver during acute inflammation. To identify the promoter element(s) involved in inducible transcription of SAA, a 5' flanking region of this gene has been fused to a reporter chloramphenicol acetyl transferase (CAT) gene and transfected into BNL liver cells. This DNA is capable of inducing synthesis of the reporter gene in response to lipopolysaccharide-stimulated monocyte-derived conditioned medium. Deletion analyses have shown that a region from -135 to -78 that contains a putative NF-kappa B element is responsible for the inducible function of the promoter. Gel shift assay has detected DNA-binding activity in the induced cells that appear to interact with the potential NF-kappa B element located within -112 to -78 of the SAA gene. Similar factor(s) have also been detected in the lipopolysaccharide-treated rabbit liver which is highly active in transcribing SAA mRNA. Appearance of these factors in acute-phase induced animals and their binding to the NF-kappa B-like element in the SAA proximal promoter region correlated with SAA mRNA synthesis suggests functional role of this promoter element in SAA gene expression under LPS-induced acute-phase condition.

A disintegrin and metalloproteinase-12 (ADAM-12), a member of multifunctional family of proteins, is upregulated in many cancers, including breast, lung, liver, prostate, gastric, and bladder. The multidomain structure, composed of a prodomain, a metalloproteinase, disintegrin-like, epidermal growth factor–like, cysteine-rich and transmembrane domains, and a cytoplasmic tail, allows ADAM-12 to promote matrix degradation, cell-cell adhesion, and intracellular signaling capacities and thereby to play a critical role in cancer growth and metastasis. Despite ample evidence linking increased ADAM-12 expression with cancer, the mechanisms controlling its upregulation are still unknown. In the present study, transforming growth factor-β1 (TGF-β1) is shown to increase ADAM-12 mRNA expression in MDA-MB-231 breast carcinoma cells. We have identified a promoter element responsible for TGF-β1–mediated ADAM-12 induction. We show interaction of NF-κB with ADAM-12 promoter and that high level of NF-κB activity in breast carcinoma cells results in the upregulation of ADAM-12 expression. Site-directed mutagenesis of the NF-κB element in ADAM-12 promoter and inhibition of NF-κB activity by Bay-11-7085 and MG-132 significantly reduced TGF-β1–mediated increase of ADAM-12 promoter-driven gene expression. Transfection of cells with a dominant-negative mutant form of IκBα (IκBαΔN), which inhibits activation of NF-κB, significantly reduced transcription from ADAM-12 promoter-reporter in TGF-β1–stimulated MDA-MB-231 cancer cells. In correlation, overexpression of NF-κB induced ADAM-12 expression in a dose-dependent manner. DNA-binding and ChIP assays indicated that p65 subunit of NF-κB binds to ADAM-12 promoter. Together, our study identified a cellular mechanism for induction of ADAM-12, which involves NF-κB and its activation by TGF-β1. Mol Cancer Res; 8(9); 1261–70. ©2010 AACR.

A genomic DNA clone that encodes serum amyloid A protein has been isolated from a rabbit liver lambda EMBL3 genomic library. Southern blot analysis indicates that the cloned gene is a member of multiple SAA gene family in rabbit. The nucleotide sequence of this DNA shows that the cloned gene corresponds to a SAA cDNA recently isolated from an acute phase rabbit liver cDNA library (1). This rabbit SAA gene contains four exons which are located within a 4500-base pair region spanning a putative TATA box and an AATAAA polyadenylation signal. DNA sequence upstream of the major transcription start site also contains other structural elements such as the sequences for NF-kappa B, IL-6 receptor and Sp1 binding. Significance of these regulatory elements in SAA gene expression is discussed.

Alpha 1-acid glycoprotein (AGP) complementary DNA clone has been isolated from an acute phase rabbit liver library. Complete and full-length nucleotide sequence of this cDNA has been determined. The cDNA contains 35 nucleotides 5' untranslated region followed by 606 nucleotides of coding region and a 112 nucleotides 3' untranslated region. Comparison of this sequence with that of human, rat and mouse reveals a high degree of homology with human alpha 1-AGP. Northern blot analysis of messenger RNA for alpha 1-AGP in normal and acute livers demonstrates remarkable induction of transcription of this gene in rabbit in response to acute inflammation by the administration of turpentine. Such a response of AGP gene expression makes it a major member of the acute phase responsive genes in rabbit liver.

A complementary DNA clone encoding serum amyloid A protein has been isolated from an acute rabbit liver cDNA library. Complete nucleotide sequence analysis reveals that the cloned gene contains a 24 bases 5′ untranslated region, 369 bases coding region and a 106 bases 3′ untraslated region. Primer extension analysis indicates that the full-length 5′ untranslated region contains 80 nucleotides. Northern blot analysis of mRNA from normal and acute rabbit livers demonstrates that this gene is expressed constitutively at a low level and undergoes induction of transcription in response to acute inflammation by the administration of turpentine.

Previously we determined that inflammation responsive transcription factors AP-1 and SAF-1 synergistically regulate transcriptional induction of the MMP-1 gene. The present study investigated the underlying molecular mechanism of cooperativity between these two different groups of transcription factors. We present evidence that knockdown of SAF-1 by small interfering RNAs inhibits AP-1-mediated increase of human MMP-1 expression. The two key members of the AP-1 family of proteins, c-Fos and c-Jun, and SAF-1 form a ternary protein complex, which has markedly higher DNA binding activity than either a SAF-1 homodimer or a c-Fos/c-Jun heterodimer. The increased DNA binding activity of the ternary complex is translated into a striking enhancement of their transcriptional activity by which synergistic transcriptional induction of MMP-1 expression is achieved. The SAF-1.c-Fos.c-Jun ternary complex efficiently promotes transcription from both SAF-1 and AP-1 sites of human MMP-1 promoter. The physical interaction between SAF-1 and AP-1 was demonstrated both in vitro by Far-Western and antibody pulldown assays with recombinant proteins and in vivo by chromatin immunoprecipitation (ChIP), re-ChIP, and co-immunoprecipitation analyses. Two distinct but adjacent domains in SAF-1 are involved in protein-protein contact with c-Fos and c-Jun; one domain resides within two N-terminal polyalanine tracts, and the other is present within the first two zinc finger motifs. Together these findings delineate the mechanism of synergy and the essential role of SAF-1 and AP-1 in up-regulating human MMP-1 expression under various inflammatory conditions.

Matrix metalloproteinase-1 (MMP-1) has been implicated in the pathogenesis of osteoarthritis (OA) due to its ability to degrade extracellular matrix component of the joint cartilage tissue that cushions the bone from frictional damage. Canine hip dysplasia, a developmental orthopedic disease which results in arthritic condition as is seen in human OA is an excellent system to study the involvement of MMP-1 in the pathogenesis of OA. To date, however, no report is available regarding canine MMP-1 promoter and the regulatory mechanism by which increased synthesis of MMP-1 protein might be regulated. To gain an insight, we have investigated the promoter region of canine MMP-1. MMP-1 synthesis in the resident cells of arthritic joints is regulated via two major cytokines, IL-1beta and TNF-alpha. By using a series of progressively deleted reporter constructs, multiple cytokine-responsive elements were identified in the proximal promoter region of canine MMP-1. These include DNA-binding elements of AP-1 and SAF-1 transcription factors. Mutation of AP-1 or SAF-1 element resulted in marked reduction in the cytokine responsiveness of MMP-1 promoter. We show that AP-1 and SAF-1 DNA-binding activities are increased in cytokine-stimulated cells as well as in osteoarthritic cartilage tissues. In correlation, immunohistochemical analysis indicated higher levels of MMP-1, SAF-1 and AP-1 proteins in osteoarthritic but not in the normal cartilage tissue. These results show that induction and activation of AP-1 and SAF-1 transcription factors are involved in the regulation of MMP-1 expression in the chondrocytes which could be used as therapeutic targets to combat pathogenesis of OA.

Based on epidemiological and pathological studies, it is becoming increasingly clear that matrix metalloproteinases (MMPs) play an important role in the pathogenesis of atherosclerosis by participating in vascular remodeling, smooth muscle cell migration, and plaque disruption. MMP-14, because of its unique ability to cause pericellular degradation, its broad substrate specificity, its synthesis in an active form, and its ability to activate other matrix metalloproteinases, is recognized as a prominent member of this family. MMP-14 is detected at high levels in the atherosclerotic plaque. To understand the induction mechanism of MMP-14 under atherogenic conditions, we examined its expression pattern in response to oxidized low-density lipoproteins (ox-LDLs) that are believed to play an important role in atherogenesis. We report that in macrophages, ox-LDLs markedly elevate the levels of MMP-14 mRNA and protein. The cis -acting elements supporting this increase were identified to be present within −213 and −1 nucleotides of the MMP-14 promoter. DNase I protection assay revealed, within this region, two major elements, of which one serves as the DNA-binding site for SAF-1 transcription factor. Increased binding of SAF-1 to the MMP-14 promoter correlated with the transcriptional upregulation of MMP-14 gene. Furthermore, induction of endogenous MMP-14 gene, MMP-14 promoter driven reporter gene expression and MMP-2 processing activity during overexpression of SAF-1 and coexpression of SAF-1 and MMP-14 in the macrophages present in the atherosclerotic plaque implicate SAF-1 as a key regulator of MMP-14 gene induction in macrophage cells.

Serum amyloid A (SAA) activating factor-1 (SAF-1) is an inducible transcription factor that plays a key role in the regulation of several inflammation-responsive genes including SAA and matrix metalloproteinase-1. Increased synthesis of SAA and matrix metalloproteinase-1 is associated with pathogenesis of several diseases including amyloidosis, arthritis, and atherosclerosis. Previously, we showed in vivo interaction of SAF-1 and protein kinase A (PKA) and presented evidence for induction of SAF-1-regulated genes by a PKA signaling pathway. Here we demonstrate a mechanism by which PKA increases functional activities of SAF-1. Site-directed mutagenesis and phosphorylation analyses revealed two sites in the SAF-1 protein, serine 187 and threonine 386, as the target of PKA. Interestingly, mutation of both PKA phosphorylation sites created a highly active SAF-1 protein with high DNA-binding ability. Furthermore, we found that terminal deletion of SAF-1 protein from either end creates SAF-1 isoforms that are highly transcriptionally active. Partial proteolysis experiments indicated that unphosphorylated and phosphorylated SAF-1 proteins are structurally distinct. Together these results suggest that under native condition, N and C termini of SAF-1 are engaged in an inhibitory intramolecular interaction. PKA-mediated phosphorylation increases transcriptional activity of SAF-1 by unmasking the DNA-binding domain.