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1.  Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm 
BMC Cell Biology  2009;10:96.
Background
Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.
Results
Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression.
Conclusions
K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.
doi:10.1186/1471-2121-10-96
PMCID: PMC2804594  PMID: 20035625
2.  An LRP5 Receptor with Internal Deletion in Hyperparathyroid Tumors with Implications for Deregulated WNT/β-Catenin Signaling 
PLoS Medicine  2007;4(11):e328.
Background
Hyperparathyroidism (HPT) is a common endocrine disorder with incompletely understood etiology, characterized by enlarged hyperactive parathyroid glands and increased serum concentrations of parathyroid hormone and ionized calcium. We have recently reported activation of the Wnt signaling pathway by accumulation of β-catenin in all analyzed parathyroid tumors from patients with primary HPT (pHPT) and in hyperplastic parathyroid glands from patients with uremia secondary to HPT (sHPT). Mechanisms that may account for this activation have not been identified, except for a few cases of β-catenin (CTNNB1) stabilizing mutation in pHPT tumors.
Methods and Findings
Reverse transcription PCR and Western blot analysis showed expression of an aberrantly spliced internally truncated WNT coreceptor low-density lipoprotein receptor–related protein 5 (LRP5) in 32 out of 37 pHPT tumors (86%) and 20 out of 20 sHPT tumors (100%). Stabilizing mutation of CTNNB1 and expression of the internally truncated LRP5 receptor was mutually exclusive. Expression of the truncated LRP5 receptor was required to maintain the nonphosphorylated active β-catenin level, transcription activity of β-catenin, MYC expression, parathyroid cell growth in vitro, and parathyroid tumor growth in a xenograft severe combined immunodeficiency (SCID) mouse model. WNT3 ligand and the internally truncated LRP5 receptor strongly activated transcription, and the internally truncated LRP5 receptor was insensitive to inhibition by DKK1.
Conclusions
The internally truncated LRP5 receptor is strongly implicated in deregulated activation of the WNT/β-catenin signaling pathway in hyperparathyroid tumors, and presents a potential target for therapeutic intervention.
Gunnar Westin and colleagues report the expression of an aberrantly spliced LRP5 receptor in primary and spontaneous parathyroid tumors and implicate it in the deregulated activation of the Wnt/β-catenin signaling pathway.
Editors' Summary
Background.
The parathyroid glands—four rice-sized glands in the neck—maintain a normal calcium balance in the body, to maintain strong bones and essential cellular functions. The glands release parathyroid hormone as a response to a decrease in blood calcium level. By stimulating calcium release from bone and its absorption in the gut, parathyroid hormone restores the blood calcium level. However, 100,000 new individuals in the US develop hyperparathyroidism (HPT) annually, characterized by enlarged, overactive parathyroid glands and high blood levels of calcium. Primary HPT (pHPT) is usually caused by a benign tumor (a non-life-threatening growth) in one of the parathyroid glands. Secondary HPT (sHPT) occurs in response to calcium regulatory disturbances, linked to vitamin D deficiency, and more or less invariably develops in patients with uremic kidney disease.
Why Was This Study Done?
HPT is usually treated by surgical removal of the enlarged parathyroid glands, which is done with great efficiency. However, ideally, doctors would like to know what drives the overgrowth of the parathyroid glands to be able to develop drugs for treatment or disease prophylaxis. Researchers recently reported that the cells in enlarged parathyroid glands from patients with HPT contain high amounts of β-catenin. This protein is part of the Wnt signaling pathway, which has been found to be disrupted in many tumor entities in other organs. In the absence of Wnt proteins, a group of proteins called the β-catenin destruction complex marks β-catenin so that it is rapidly destroyed. When Wnt proteins bind to a cell-surface receptor called Frizzled and a coreceptor called LRP5, the destruction complex is inhibited and β-catenin accumulates. This accumulation induces the production of other proteins (in particular, c-Myc) that stimulate cell growth and division. The accumulation of β-catenin in the enlarged parathyroid glands of patients with HPT could, therefore, significantly contribute to the overgrowth of their glands—but what causes β-catenin accumulation? In this study, the researchers have investigated this question to try to identify a target for drugs to treat HPT.
What Did the Researchers Do and Find?
The researchers looked for genetic changes (mutations) in β-catenin that stabilize the protein and measured the expression of LRP5 in abnormal parathyroid gland tissue from 37 patients with pHPT and 20 with uremia and sHPT. All the samples contained high levels of β-catenin, but only four contained a β-catenin–stabilizing mutation. All the sHPT samples and 32 pHPT samples (but none of the samples containing the β-catenin stabilizing mutation) expressed a mutated LRP5, with the central region deleted. To investigate the functional consequences of this internally deleted LRP5 protein, the researchers used a technique called RNA interference to block its expression in a human parathyroid tumor cell line. They found that expression of the mutated, short LRP5 is required for accumulation of β-catenin, expression of c-Myc, and continued growth of the cell line in test tubes and in animals.
What Do These Findings Mean?
The accumulation of β-catenin in all the enlarged parathyroid glands examined so far strongly implicates abnormal Wnt/β-catenin signaling in the development of pHPT and sHPT. These new findings identify which part of the signaling pathway is altered. The expression data and functional data together suggest that an internally deleted LRP5 coreceptor is often responsible for the accumulation of β-catenin. The functional data also show that expression of shortened LRP5 is necessary for the abnormal growth of parathyroid tumor cells. Exactly how the internally deleted coreceptor activates β-catenin signaling in parathyroid gland cells, or why a shorter-than-normal LRP5 is made, are not yet known. However, because these findings indicate that internally deleted LRP5 has a fundamental role in activating Wnt signaling in HPT, drugs that inactivate this aberrant protein but leave the normal protein unscathed might provide a nonsurgical treatment for this common hormone disorder.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040328.
edlinePlus has encyclopedia pages on hyperparathyroidism, primary hyperparathyroidism, and secondary hyperparathyroidim (in English and Spanish)
Information is available for patients from the US National Institute of Diabetes and Digestive and Kidney Diseases on hyperparathyroidism, which includes links to organizations that help people with hyperparathyroidism
Wikipedia maintains pages on Wnt signaling pathway and on β-catenin (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0040328
PMCID: PMC2082644  PMID: 18044981
3.  Dissecting Molecular Differences between Wnt Coreceptors LRP5 and LRP6 
PLoS ONE  2011;6(8):e23537.
Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening “gap4” region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.
doi:10.1371/journal.pone.0023537
PMCID: PMC3160902  PMID: 21887268
4.  Differential Modulation of TCF/LEF-1 Activity by the Soluble LRP6-ICD 
PLoS ONE  2010;5(7):e11821.
The canonical Wnt/β-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions.
doi:10.1371/journal.pone.0011821
PMCID: PMC2911377  PMID: 20676368
5.  Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis 
Oncogene  2011;31(26):3202-3212.
The aspartic protease cathepsin-D (cath-D) is a marker of poor prognosis in breast cancer that is overexpressed and hypersecreted by human breast cancer cells. Secreted pro-cath-D binds to the extracellular domain of the β chain of the LDL receptor-related protein-1 (LRP1) in fibroblasts. The LRP1 receptor has an 85-kDa transmembrane β chain and a non-covalently attached 515-kDa extracellular α chain. LRP1 acts by (1) internalizing many ligands via its α chain, (2) activating signaling pathways by phosphorylating the LRP1β chain tyrosine, and (3) modulating gene transcription by regulated intramembrane proteolysis (RIP) of its β chain. LRP1 RIP involves two cleavages: the first liberates the LRP1 ectodomain to give a membrane-associated form LRP1β-CTF and the second generates the LRP1β intracellular domain, LRP1β-ICD, that modulates gene transcription. Here, we investigated the endocytosis of pro-cath-D by LRP1 and the effect of the pro-cath-D/LRP1β interaction on LRP1β tyrosine phosphorylation and/or LRP1β RIP. Our results indicate that pro-cath-D was partially endocytosed by LRP1 in fibroblasts. However, pro-cath-D and ectopic cath-D did not stimulate phosphorylation of the LRP1β chain tyrosine. Interestingly, ectopic cath-D and its catalytically-inactive D231Ncath-D, and pro-D231Ncath-D all significantly inhibited LRP1 RIP by preventing LRP1β-CTF production. Thus cath-D inhibits LRP1 RIP independently of its catalytic activity by blocking the first cleavage. Since cath-D triggers fibroblast outgrowth via LRP1, we propose that cath-D modulates the growth of fibroblasts by inhibiting LRP1 RIP in the breast tumor micro-environment.
doi:10.1038/onc.2011.501
PMCID: PMC3579766  PMID: 22081071
Animals; Breast Neoplasms; metabolism; pathology; COS Cells; Cathepsin D; metabolism; Cell Line, Tumor; Cell Membrane; metabolism; Cell Proliferation; Cercopithecus aethiops; Endocytosis; Enzyme Precursors; metabolism; Fibroblasts; cytology; enzymology; metabolism; pathology; Humans; Low Density Lipoprotein Receptor-Related Protein-1; chemistry; metabolism; Mammary Glands, Human; cytology; pathology; Neoplasm Invasiveness; Protein Structure, Tertiary; Proteolysis; Tumor Microenvironment; cancer; cathepsin D; LRP1; RIP; endocytosis; tyrosine phosphorylation
6.  Low-density lipoprotein receptor–related protein 5 governs Wnt-mediated osteoarthritic cartilage destruction 
Introduction
Wnt ligands bind to low-density lipoprotein receptor–related protein (LRP) 5 or 6, triggering a cascade of downstream events that include β-catenin signaling. Here we explored the roles of LRP5 in interleukin 1β (IL-1β)- or Wnt-mediated osteoarthritic (OA) cartilage destruction in mice.
Methods
The expression levels of LRP5, type II collagen, and catabolic factors were determined in mouse articular chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild-type, Lrp5 total knockout (Lrp5-/-) and chondrocyte-specific knockout (Lrp5fl/fl;Col2a1-cre) mice was caused by aging, destabilization of the medial meniscus (DMM), or intra-articular injection of collagenase. The role of LRP5 was confirmed in vitro by small interfering RNA–mediated knockdown of Lrp5 or in Lrp5-/- cells treated with IL-1β or Wnt proteins.
Results
IL-1β treatment increased the expression of LRP5 (but not LRP6) via JNK and NF-κB signaling. LRP5 was upregulated in human and mouse OA cartilage, and Lrp5 deficiency in mice inhibited cartilage destruction. Treatment with IL-1β or Wnt decreased the level of Col2a1 and increased those of Mmp3 or Mmp13, whereas Lrp5 knockdown ameliorated these effects. In addition, we found that the functions of LRP5 in arthritic cartilage were subject to transcriptional activation by β-catenin. Moreover, Lrp5-/- and Lrp5fl/fl;Col2a1-cre mice exhibited decreased cartilage destruction (and related changes in gene expression) in response to experimental OA.
Conclusions
Our findings indicate that LRP5 (but not LRP6) plays an essential role in Wnt/β-catenin-signaling-mediated OA cartilage destruction in part by regulating the expression levels of type II collagen, MMP3, and MMP13.
doi:10.1186/ar4466
PMCID: PMC3978879  PMID: 24479426
7.  Effects of nutrition and growth rate on Lrp levels in Escherichia coli. 
Journal of Bacteriology  1996;178(23):6930-6936.
Lrp (leucine-responsive regulatory protein) activates some Escherichia coli operons that function in anabolism and represses others involved in catabolism (for a review, see J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). This overall pattern suggests that Lrp may help cells adapt to changes in the nutritional environment. Here, we tested the idea that the nutritional richness of the medium determines the amount of Lrp in cells. Lrp was measured directly by Western blotting (immunoblotting) in cells grown in a chemically defined rich medium or in a minimal medium. In addition, transcription from the lrp promoter was assessed with a lacZ reporter gene. The results with these two different measurements were nearly the same, indicating that under the conditions employed, beta-galactosidase measurements can accurately reflect Lrp levels. For cells in a minimal medium, Lrp levels were consistently lowest during the logarithmic phase of growth, but overall, there was not much variation in levels as a function of growth phase (1.3-fold difference between highest and lowest values). However, for cells in a rich medium, Lrp levels dropped 3- to 4-fold during the lag phase, remained constant during the log phase, and then rose to starting levels upon entry into the stationary phase. When cells in the log phase were compared, Lrp levels were 3- to 4-fold higher in cells growing in a minimal medium than those in a rich medium. The levels of lrp expression were the same or slightly higher in strains containing mutations in rpoS, cya, or crp compared with wild-type strains, suggesting that neither RpoS nor the cyclic AMP (cAMP) receptor protein-cAMP complex is required for expression. On the other hand, lrp expression was severely restricted in cells that could not make ppGpp because of mutations in relA and spoT. The reduced expression of lrp during logarithmic growth in a rich medium may be due to low ppGpp levels under these conditions. The repressive effects of rich medium and the stimulatory effects of ppGpp were also observed with a construct having only a minimal lrp promoter (-57 to +21). The results of other experiments suggest that Lrp levels vary inversely with the growth rate of cells instead of being determined by some component of the medium.
PMCID: PMC178595  PMID: 8955316
8.  Low Level of Low-Density Lipoprotein Receptor-Related Protein 1 Predicts an Unfavorable Prognosis of Hepatocellular Carcinoma after Curative Resection 
PLoS ONE  2012;7(3):e32775.
Background
Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC).
Methods
LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens.
Results
Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set.
Conclusions
LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.
doi:10.1371/journal.pone.0032775
PMCID: PMC3299691  PMID: 22427881
9.  Lrp Acts as Both a Positive and Negative Regulator for Type 1 Fimbriae Production in Salmonella enterica Serovar Typhimurium 
PLoS ONE  2011;6(10):e26896.
Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrpC) mutant strains. In this work, we used chromosomal PfimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to PfimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in PfimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to PfimA without competitive exclusion. In addition, both Lrp and FimZ binding to PfimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.
doi:10.1371/journal.pone.0026896
PMCID: PMC3203922  PMID: 22046399
10.  Bone morphogenetic protein-2-induced Wnt/β-catenin signaling pathway activation through enhanced low-density-lipoprotein receptor-related protein 5 catabolic activity contributes to hypertrophy in osteoarthritic chondrocytes 
Introduction
Events normally taking place in the terminal chondrocyte differentiation in the growth plate are also observed during osteoarthritis (OA) development, suggesting that molecules, such as Wnts and bone morphogenetic proteins (BMPs) regulating chondrocyte activity in the growth plate, may play a key role in osteoarthritis pathogenesis. The aim of the study was to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in OA progression.
Methods
Low-density-lipoprotein receptor-related protein 5 (LRP-5) and 6, BMP-2, -4, and -7, bone morphogenetic protein receptor-IA and IB (BMPR-IA and BMPR-IA), lymphoid enhancer factor-1 (LEF-1), and transcription factor 4 (TCF-4) expression levels were investigated in normal and osteoarthritic chondrocytes. LRP-5, β-catenin (phospho and active form), matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, as well as collagen X (COL10A1) expression levels were evaluated after LRP-5 silencing in BMP-2-treated chondrocytes. The investigation of Smad1/5/8 binding to LRP-5 promoter was assessed with chromatin immunoprecipitation (ChIP). Furthermore, we evaluated the effect of experimental activation of the Wnt/β-catenin pathway with LiCl and LEF-1 silencing, in LiCl-treated chondrocytes, on matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, and collagen X (COL10A1) expression, as well as possible interactions between LEF-1 and MMPs and COL10A1 promoters by using a ChIP assay.
Results
LRP-5, BMP-2, BMP-4, BMPR-IA, and LEF-1 mRNA and protein expression levels were found to be significantly upregulated in osteoarthritic chondrocytes compared with normal. We showed that treatment of cultured chondrocytes with BMP-2 resulted in increased β-catenin nuclear translocation and LRP-5 expression and that the BMP-2-induced LRP-5 upregulation is mediated through Smad1/5/8 binding on LRP-5 promoter. LRP-5 silencing reduced nuclear β-catenin protein levels, MMPs and collagen X expression, whereas increased phospho-β-catenin protein levels in BMP-2-treated chondrocyte. Furthermore, we demonstrated that activation of the Wnt/β-catenin signaling pathway by LiCl and LEF-1 downregulation by using siRNA regulates MMP-9, 13, 14, ADAMTS-5, and COL10A1 expression, evidenced by the observed strong binding of LEF-1 to MMP-9, 13, 14, ADAMTS-5 and COL10A promoters.
Conclusions
Our findings suggest, for the first time to our knowledge, that BMP-2-induced Wnt/β-catenin signaling activation through LRP-5 may contribute to chondrocyte hypertrophy and cartilage degradation in osteoarthritis.
doi:10.1186/ar3805
PMCID: PMC3446456  PMID: 22513174
11.  Macrophage LRP1 Suppresses Neo-Intima Formation during Vascular Remodeling by Modulating the TGF-β Signaling Pathway 
PLoS ONE  2011;6(12):e28846.
Background
Vascular remodeling in response to alterations in blood flow has been shown to modulate the formation of neo-intima. This process results from a proliferative response of vascular smooth muscle cells and is influenced by macrophages, which potentiate the development of the intima. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that recognizes a number of ligands including apoE-containing lipoproteins, proteases and protease-inhibitor complexes. Macrophage LRP1 is known to influence the development of atherosclerosis, but its role in vascular remodeling has not been investigated.
Methodology/Principal Findings
To define the contribution of macrophage LRP1 to vascular remodeling, we generated macrophage specific LRP1-deficient mice (macLRP1-/-) on an LDL receptor (LDLr) knock-out background. Using a carotid ligation model, we detected a 2-fold increase in neointimal thickening and a 2-fold increase in the intima/media ratio in macLRP1-/- mice. Quantitative RT-PCR arrays of the remodeled vessel wall identified increases in mRNA levels of the TGF-β2 gene as well as the Pdgfa gene in macLRP1-/- mice which could account for the alterations in vascular remodeling. Immunohistochemistry analysis revealed increased activation of the TGF-β signaling pathway in macLRP1-/- mice. Further, we observed that LRP1 binds TGF-β2 and macrophages lacking LRP1 accumulate twice as much TGF-β2 in conditioned media. Finally, TNF-α modulation of the TGF-β2 gene in macrophages is attenuated when LRP1 is expressed. Together, the data reveal that LRP1 modulates both the expression and protein levels of TGF-β2 in macrophages.
Conclusions/Significance
Our data demonstrate that macrophage LRP1 protects the vasculature by limiting remodeling events associated with flow. This appears to occur by the ability of macrophage LRP1 to reduce TGF-β2 protein levels and to attenuate expression of the TGF-β2 gene resulting in suppression of the TGF-β signaling pathway.
doi:10.1371/journal.pone.0028846
PMCID: PMC3235159  PMID: 22174911
12.  The Internally Truncated LRP5 Receptor Presents a Therapeutic Target in Breast Cancer 
PLoS ONE  2009;4(1):e4243.
Background
Breast cancer is a common malignant disease, which may be caused by a number of genes deregulated by genomic or epigenomic events. Deregulated WNT/β-catenin signaling with accumulation of β-catenin is common in breast tumors, but mutations in WNT signaling pathway components have been rare. An aberrantly spliced internally truncated LRP5 receptor (LRP5Δ666–809, LRP5Δ) was shown recently to be resistant to DKK1 inhibition, and was required for β-catenin accumulation in hyperparathyroid tumors and parathyroid tumor growth.
Methodology/Principal Findings
Here we show, by reverse transcription PCR and Western blot analysis, that LRP5Δ is frequently expressed in breast tumors of different cancer stage (58–100%), including carcinoma in situ and metastatic carcinoma. LRP5Δ was required in MCF7 breast cancer cells for the non-phosphorylated active β-catenin level, transcription activity of β-catenin, cell growth in vitro, and breast tumor growth in a xenograft SCID mouse model. WNT3 ligand, but not WNT1 and WNT3A augmented the endogenous β-catenin activity of MCF7 cells in a DKK1-insensitive manner. Furthermore, an anti-LRP5 antibody attenuated β-catenin activity, inhibited cell growth, and induced apoptosis in LRP5Δ-positive MCF7 and T-47D breast cancer cells, but not in control cells.
Conclusions/Significance
Our results suggest that the LRP5Δ receptor is strongly implicated in mammary gland tumorigenesis and that its aberrant expression present an early event during disease progression. LRP5 antibody therapy may have a significant role in the treatment of breast cancer.
doi:10.1371/journal.pone.0004243
PMCID: PMC2627768  PMID: 19158955
13.  Specific binding of PapI to Lrp-pap DNA complexes. 
Journal of Bacteriology  1995;177(22):6449-6455.
Expression of pyelonephritis-associated pili (Pap) varies between transcriptionally active (ON) and inactive (OFF) phase states. Pap phase variation is controlled by the binding of leucine-responsive regulatory protein (Lrp) to two pap regulatory DNA regions, each containing a deoxyadenosine methylase site and designated GATC-I and GATC-II. Methylation of these GATC sites modulates binding of Lrp and plays an essential role in phase variation. PapI, an 8.8-kDa pap-encoded regulatory protein, plays a key role in the switch between OFF and ON transcription states. In the absence of PapI, Lrp binds to sites overlapping the papBA promoter and inhibits transcription. Addition of PapI results in a translocation of Lrp binding to sites over 100 bp upstream, resulting in the ON transcription state. Gel shift analysis using radiolabeled PapI shows that PapI binds with high specificity to Lrp-pap DNA complexes but binds only weakly to free Lrp. Protein cross-linking studies indicate that Lrp and PapI directly interact with each other. On the basis of these data, we present a hypothesis in which PapI facilitates the transition between OFF and ON transcription states by binding to Lrp and altering Lrp's affinity for the pap GATC-I and GATC-II regions.
PMCID: PMC177494  PMID: 7592419
14.  Helicobacter pylori-induced activation of β-catenin involves low density lipoprotein receptor-related protein 6 and Dishevelled 
Molecular Cancer  2010;9:31.
Background
The human microbial pathogen Helicobacter pylori resides in the stomach of about fifty percent of the world's population and represents a risk factor for chronic gastritis, peptic ulcers and, in rare cases, gastric cancer. Alterations of the Wnt/β-catenin signaling pathway have been described in almost every human cancer disease, due to the regulation of target genes being involved in cell cycle control, differentiation, cell migration or stem cell control. Our study aimed to elucidate the role of proximal Wnt signaling components low density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled (Dvl) in the activation of β-catenin early after infection of gastric epithelial cells with H. pylori.
Results
Infection of gastric epithelial NCI-N87 cells with H. pylori induces rapid phosphorylation of the Wnt/β-catenin pathway co-receptor LRP6 independent of the cytotoxin-associated gene A (CagA) or vacuolating cytotoxin A (VacA). However, bacteria lacking a functional type 4 secretion system (T4SS) failed to induce LRP6 phosphorylation. Further, we identified proteins of the Dvl family, namely Dvl2 and Dvl3, which are involved in LRP6 phosphorylation. H. pylori-induced nuclear accumulation of β-catenin and its transcriptional activation, and expression of Wnt target genes are strongly reduced in stable knockdown cell lines deficient for LRP6, Dvl2 or Dvl3.
Conclusion
We analysed the H. pylori-induced activation of Wnt-signaling factors and demonstrate for the first time that the canonical Wnt-signaling proteins LRP6 and Dvl2 and Dvl3 are involved in the regulation of β-catenin.
doi:10.1186/1476-4598-9-31
PMCID: PMC2825249  PMID: 20137080
15.  Leucine-Responsive Regulatory Protein (Lrp) Acts as a Virulence Repressor in Salmonella enterica Serovar Typhimurium▿  
Journal of Bacteriology  2008;191(4):1278-1292.
Leucine-responsive regulatory protein (Lrp) is a global gene regulator that influences expression of a large number of genes including virulence-related genes in Escherichia coli and Salmonella. No systematic studies examining the regulation of virulence genes by Lrp have been reported in Salmonella. We report here that constitutive expression of Lrp [lrp(Con)] dramatically attenuates Salmonella virulence while an lrp deletion (Δlrp) mutation enhances virulence. The lrp(Con) mutant caused pleiotropic effects that include defects in invasion, cytotoxicity, and colonization, whereas the Δlrp mutant was more proficient at these activities than the wild-type strain. We present evidence that Lrp represses transcription of key virulence regulator genes—hilA, invF, and ssrA—in Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2), by binding directly to their promoter regions, PhilA, PinvF, and PssrA. In addition, Western blot analysis showed that the expression of the SPI-1 effector SipA was reduced in the lrp(Con) mutant and enhanced in the Δlrp mutant. Computational analysis revealed putative Lrp-binding consensus DNA motifs located in PhilA, PinvF, and PssrA. These results suggest that Lrp binds to the consensus motifs and modulates expression of the linked genes. The presence of leucine enhanced Lrp binding to PinvF in vitro and the addition of leucine to growth medium decreased the level of invF transcription. However, leucine had no effect on expression of hilA and ssrA or on cellular levels of Lrp. In addition, Lrp appears to be an antivirulence gene, since the deletion mutant showed enhanced cell invasion, cytotoxicity, and hypervirulence in BALB/c mice.
doi:10.1128/JB.01142-08
PMCID: PMC2631999  PMID: 19074398
16.  Lrp is a direct repressor of the dad operon in Escherichia coli. 
Journal of Bacteriology  1996;178(24):7234-7240.
Expression of the degradative D-amino acid dehydrogenase (dad) operon is known to be increased when Escherichia coli is grown in the presence of D- or L-alanine. Alanine is thought to act as an inducer to block the action of a postulated repressor. This operon is also believed to be regulated by catabolite repression. We have used in vivo and in vitro experiments that show that the dad repressor is the leucine-responsive regulatory protein (Lrp). dad expression in a dad-lacZ operon fusion strain was increased four- to sevenfold when cells were grown in minimal medium containing alanine or leucine. A strain lacking Lrp had high-level constitutive dad expression. Gel retardation and footprinting studies revealed that Lrp binds in vitro to multiple sites over a large area in the dad promoter region. This binding was reduced by alanine or leucine. In vitro transcription assays, using a plasmid template and primer extension analysis, identified three major dad transcripts (Tr1, Tr2, and Tr3). The formation of these transcripts was differentially regulated by cyclic AMP-cyclic AMP receptor protein complex, and each was strongly repressed by Lrp. Alanine or leucine completely (for Tr1 and Tr2) or partially (for Tr3) reversed Lrp inhibition. Site-directed mutagenesis of an Lrp binding site strongly reduced Lrp binding and prevented Lrp repression of dad transcription in vivo and in vitro. Taken together, these results strongly suggest that Lrp and alanine or leucine act directly to repress and induce, respectively, transcription of the dad operon.
PMCID: PMC178638  PMID: 8955407
17.  The Unfolded Protein Response is a Major Mechanism by which LRP1 Regulates Schwann Cell Survival After Injury 
In peripheral nerve injury, Schwann cells (SCs) must survive to exert a continuing and essential role in successful nerve regeneration. Herein, we show that peripheral nerve injury is associated with activation of endoplasmic reticulum (ER) stress and the adaptive unfolded protein response (UPR). The UPR culminates in expression of C/EBP homology protein CHOP, a pro-apoptotic transcription factor in SCs, unless counteracted by LDL receptor-related protein-1 (LRP1), which serves as a major activator of phosphatidylinositol 3-kinase (PI3K). Sciatic nerve crush injury in rats induced expression of the ER chaperone, GRP78/BIP, reflecting an early, corrective phase of the UPR. However, when LRP1-signaling was inhibited with receptor-associated protein, PI3K activity was decreased and CHOP protein expression increased, particularly in myelinating SCs. In cultured SCs, the PKR-like ER kinase (PERK) target, eIF2α, was phosphorylated and CHOP was induced by: 1) inhibiting PI3K; 2) treating the cells with tumor necrosis factor-α (TNF-α); or 3) genetic silencing of LRP1. CHOP gene deletion in SCs decreased cell death in response to TNF-α. Furthermore, the effects of TNF-α on p-eIF2α, CHOP, and SC death were blocked by adding LRP1 ligands that augment LRP1-dependent cell-signaling to PI3K. Collectively, our results support a model in which UPR-activated signaling pathways represent a major challenge to SC survival in nerve injury. LRP1 functions as a potent activator of PI3K in SCs and by this mechanism, limits SC apoptosis resulting from increased CHOP expression in nerve injury.
doi:10.1523/JNEUROSCI.2850-11.2011
PMCID: PMC3188465  PMID: 21940431
Schwann cell; peripheral nerve regeneration; ER stress; CHOP; survival; phosphatidylinositol 3-kinase (PI3K)
18.  LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure 
Human Molecular Genetics  2013;22(21):4267-4281.
Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulation were examined using gain (Crooked tail, Lrp6Cd) and loss (Lrp6−) of function mouse lines. Two features often associated with canonical Wnt signaling, dorsal–ventral patterning and proliferation, were no different from wild-type (WT) in the Lrp6Cd/Cd neural tube. Lrp6−/− embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6Cd/Cd and Lrp6−/− cranial folds were indicated by cell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6Cd/Cd or Lrp6−/− embryos exhibited elevated and decreased RhoA basal activity levels, respectively. While ligand-independent activation of canonical Wnt signaling, bypassing Lrp-Frizzled receptors, did not activate RhoA, non-canonical Wnt5a stimulation of RhoA activity was impaired in Lrp6−/− MEFs. RhoA inhibition exacerbated NTDs in cultured Lrp6 knockout embryos compared with WT littermates. In contrast, a ROCK inhibitor rescued Lrp6Cd/Cd embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity in Wnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6Cd protein where interaction with DAAM1 could account for observed elevated RhoA activity. Conversely, null mutation that eliminates Lrp6 interaction with DAAM1 led to lower basal RhoA activity in Lrp6−/− embryos. These results indicate that Lrp6 mediates not only canonical Wnt signaling, but can also modulate non-canonical pathways involving RhoA-dependent mechanisms to impact neurulation, possibly through intracellular complexes with DAAM1.
doi:10.1093/hmg/ddt277
PMCID: PMC3792688  PMID: 23773994
19.  Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein? 
Journal of Bacteriology  1993;175(22):7160-7169.
The regulon controlled by the leucine-responsive regulatory protein (Lrp) of Escherichia coli consists of over 40 genes and proteins whose expression is regulated, either positively or negatively, by Lrp. The gltBDF operon, encoding glutamate synthase, was originally identified as a member of the Lrp regulon through a two-dimensional electrophoretic analysis of polypeptides from isogenic strains containing or lacking a functional Lrp protein. We have now demonstrated that Lrp regulates the transcription of gltBDF::lacZ operon fusions. Relative to expression in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium, gltBDF::lacZ expression in an lrp+ strain is repressed 2.2-fold in the presence of 10 mM exogenous leucine and 16-fold in Luria broth. Repression of gltBDF::lacZ expression by leucine or Luria broth is not seen for an isogenic strain containing a Tn10 insertion in lrp, and expression of gltBDF::lacZ is 44-fold lower than in the lrp+ strain when both are grown in glucose minimal MOPS medium. Lrp binds specifically to DNA fragments containing the gltBDF promoter region. Saturating levels of leucine do not abolish binding of Lrp upstream of gltBDF but merely increase its apparent dissociation constant from 2.0 to 6.9 nM. Electrophoretic analysis of the Lrp regulon established that target proteins differ greatly in the degree to which the effect of Lrp on their expression is antagonized by leucine. On the basis of our present results, we present a model for positive regulation of target genes by Lrp. Insensitivity to leucine would be expected when the effective intracellular concentration of Lrp is high relative to the affinity of Lrp binding sites required for transcription of the target gene. At lower concentrations of Lrp, transcription of the target gene should be sensitive to leucine. This model suggests that regulation of the concentration of active Lrp is critical to control of the Lrp regulon.
Images
PMCID: PMC206857  PMID: 7901196
20.  Regulation of Cytokine Expression by Schwann Cells in Response to α2-Macroglobulin Binding to LRP1 
Journal of neuroscience research  2011;89(4):544-551.
Binding of activated α2-macroglobulin (α2M) to LDL receptor-related protein-1 (LRP1) in Schwann cells activates ERK/MAP kinase and Akt and thereby promotes cell survival and migration. The goal of this study was to determine whether α2M binding to LRP1 regulates expression of cytokines and chemokines. To assess the LRP1 response selectively, we studied primary cultures of rat Schwann cells. In a screening assay that detects 84 gene products, monocyte chemoattractant protein-1 (MCP-1/CCL2) mRNA expression was increased more than 13-fold in Schwann cells treated with activated α2M. The effects of α2M on MCP-1 expression were selective, because expression of the general proinflammatory cytokine tumor necrosis factor-α (TNF-α) was not induced. We confirmed that α2M selectively induces expression of MCP-1 and not TNF-α in single-target qPCR assays. MCP-1 protein accumulated at increased levels in conditioned medium of α2M-treated cells. LRP1 was necessary for induction of MCP-1 expression, as determined in experiments with the LRP1 antagonist receptor-associated protein, a mutated form of full-length α2M that does not bind LRP1, and in studies with Schwann cells in which LRP1 was silenced. Inhibiting ERK/MAP kinase activation blocked expression of MCP-1. These studies support a model in which LRP1 regulates multiple aspects of Schwann cell physiology in the response to PNS injury.
doi:10.1002/jnr.22576
PMCID: PMC3560970  PMID: 21290408
α2-macroglobulin; LDL receptor-related protein-1; monocyte chemoattractant protein-1; TNF-α; Schwann cell; protease
21.  Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli. 
Journal of Bacteriology  1992;174(4):1109-1118.
The leucine-responsive regulatory protein (Lrp) has been shown to regulate, either positively or negatively, the transcription of several Escherichia coli genes in response to leucine. We have used two-dimensional gel electrophoresis to analyze the patterns of polypeptide expression in isogenic lrp+ and lrp mutant strains in the presence or absence of leucine. The absence of a functional Lrp protein alters the expression of at least 30 polypeptides. The expression of the majority of these polypeptides is not affected by the presence or absence of 10 mM exogenous leucine. Outer membrane porins OmpC and OmpF, glutamine synthetase (GlnA), the small subunit of glutamate synthase (GltD), lysyl-tRNA synthetase form II (LysU), a high-affinity periplasmic binding protein specific for branched-chain amino acids (LivJ), W protein, and the enzymes of the pathway converting threonine to glycine, namely, threonine dehydrogenase (Tdh) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), were identified as members of the Lrp regulon by electrophoretic analysis. We have shown that Lrp is a positive regulator of glutamate synthase and glutamine synthetase and that exogenous leucine has little or no effect on the expression of these proteins. In strains carrying a glnL deletion and in strains carrying the glnL2302 allele, which directs the synthesis of a GlnL protein that is constitutively active, expression of glutamine synthetase is no longer regulated by Lrp, demonstrating that the effect of Lrp on glutamine synthetase levels is indirect and requires an intact glnL gene. lrp::Tn10 strains grow poorly when arginine or ornithine is present as the sole nitrogen source in the medium. On the bases of present studies and previous research, we propose that Lrp is involved in the adaptation of E. coli cells to major shifts in environment, such as those which occur when E. coli leaves the intestinal tract of its animal host. Several genes required for amino acid and peptide transport and catabolism are negatively regulated by Lrp, and other genes required for amino acid biosynthesis and ammonia assimilation in a nitrogen-poor environment are positively regulated by Lrp.
Images
PMCID: PMC206403  PMID: 1346534
22.  A shed form of LDL receptor–related protein–1 regulates peripheral nerve injury and neuropathic pain in rodents 
Injury to the peripheral nervous system (PNS) initiates a response controlled by multiple extracellular mediators, many of which contribute to the development of neuropathic pain. Schwann cells in an injured nerve demonstrate increased expression of LDL receptor–related protein–1 (LRP1), an endocytic receptor for diverse ligands and a cell survival factor. Here we report that a fragment of LRP1, in which a soluble or shed form of LRP1 with an intact α-chain (sLRP-α), was shed by Schwann cells in vitro and in the PNS after injury. Injection of purified sLRP-α into mouse sciatic nerves prior to chronic constriction injury (CCI) inhibited p38 MAPK activation (P-p38) and decreased expression of TNF-α and IL-1β locally. sLRP-α also inhibited CCI-induced spontaneous neuropathic pain and decreased inflammatory cytokine expression in the spinal dorsal horn, where neuropathic pain processing occurs. In cultures of Schwann cells, astrocytes, and microglia, sLRP-α inhibited TNF-α–induced activation of p38 MAPK and ERK/MAPK. The activity of sLRP-α did not involve TNF-α binding, but rather glial cell preconditioning, so that the subsequent response to TNF-α was inhibited. Our results show that sLRP-α is biologically active and may attenuate neuropathic pain. In the PNS, the function of LRP1 may reflect the integrated activities of the membrane-anchored and shed forms of LRP1.
doi:10.1172/JCI32371
PMCID: PMC2104478  PMID: 18060043
23.  Upregulation of the Wnt Co-Receptor LRP6 Promotes Hepatocarcinogenesis and Enhances Cell Invasion 
PLoS ONE  2012;7(5):e36565.
Background
Activation of the Wnt/β-catenin signaling pathway plays a crucial role in hepatocellular carcinoma (HCC). Low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) is one of the co-receptors of the Wnt/β-catenin pathway and forms a signaling complex with Wnt ligand and Frizzled receptor to activate downstream signaling. However, the role of LRP6 in hepatocarcinogenesis is unclear. In this study, we examined its expression and roles in human HCC.
Methodology/Principal Findings
Using real-time quantitative RT-PCR, we found that LRP6 was frequently (45%) overexpressed in human HCCs (P = 0.003). In vitro studies showed that ectopic expression of LRP6 increased the protein level of β-catenin. Moreover, overexpression of the full-length and constitutively active LRP6, respectively, activated the WNT/β-catenin signaling pathway, as shown by the TCF/β-catenin reporter assay. With regard to the effects of LRP6 overexpression in HCC cells, stable overexpression of the constitutively active LRP6 in BEL-7402 HCC cells enhanced cell proliferation, cell migration, and invasion in vitro as well as tumorigenicity in nude mice.
Conclusions/Significance
Our findings indicate that overexpression of LRP6 contributes to the hyperactivation of the Wnt/β-catenin signaling pathway in human HCCs and suggest it may play a role in hepatocarcinogenesis.
doi:10.1371/journal.pone.0036565
PMCID: PMC3343020  PMID: 22570728
24.  UXT is a novel and essential cofactor in the NF-κB transcriptional enhanceosome 
The Journal of Cell Biology  2007;178(2):231-244.
As a latent transcription factor, nuclear factor κB (NF-κB) translocates from the cytoplasm into the nucleus upon stimulation and mediates the expression of genes that are important in immunity, inflammation, and development. However, little is known about how it is regulated inside the nucleus. By a two-hybrid approach, we identify a prefoldin-like protein, ubiquitously expressed transcript (UXT), that is expressed predominantly and interacts specifically with NF-κB inside the nucleus. RNA interference knockdown of UXT leads to impaired NF-κB activity and dramatically attenuates the expression of NF-κB–dependent genes. This interference also sensitizes cells to apoptosis by tumor necrosis factor-α. Furthermore, UXT forms a dynamic complex with NF-κB and is recruited to the NF-κB enhanceosome upon stimulation. Interestingly, the UXT protein level correlates with constitutive NF-κB activity in human prostate cancer cell lines. The presence of NF-κB within the nucleus of stimulated or constitutively active cells is considerably diminished with decreased endogenous UXT levels. Our results reveal that UXT is an integral component of the NF-κB enhanceosome and is essential for its nuclear function, which uncovers a new mechanism of NF-κB regulation.
doi:10.1083/jcb.200611081
PMCID: PMC2064443  PMID: 17620405
25.  ilvIH Operon Expression in Escherichia coli Requires Lrp Binding to Two Distinct Regions of DNA 
Journal of Bacteriology  2002;184(19):5293-5300.
The leucine-responsive regulatory protein Lrp regulates the expression of a number of operons in Escherichia coli, including the ilvIH operon. Earlier in vitro experiments showed purified Lrp binding to two regions of DNA proximal to the ilvIH promoter, an upstream region (−260 to −190) and a downstream region (−150 to −40). The effect of mutations in these regions on ilvIH promoter expression in vivo led to the proposal that activation of transcription required Lrp binding to downstream sites 3, 4, 5, and 6. Binding of Lrp to upstream sites 1 and 2 seemed to enhance promoter expression but was not absolutely required (Q. Wang and J. M. Calvo, J. Mol. Biol. 229:306-318, 1993). Here we present data that require a reevaluation of the above conclusion. Constructs having either a deletion of DNA or a 100-bp substitution of DNA upstream of position −160 showed no ilvIH promoter activity in vivo. These results unambiguously establish that DNA at or upstream of position −160 is required for ilvIH promoter expression. Together with previous results, we conclude that Lrp bound at downstream sites is necessary but not sufficient for promoter activation. In addition, insertion of 4, 6, 8, or 10 bp between the upstream and downstream regions also resulted in a very strong reduction of in vivo promoter expression, even though the binding of Lrp in vitro was not greatly affected by these mutations. Closer inspection showed that the affinity of Lrp for the upstream region of all of these constructs was about the same but that Lrp bound to the downstream region of the wild-type construct with a higher degree of cooperativity than in the case of the others. These mutations may have reduced promoter activity in vivo by eliminating a binding site for some transcription factor other than Lrp. Alternatively, the small-addition mutations may have affected the geometry of these complexes, preventing either an interaction between Lrps bound at upstream and downstream sites (which might be necessary for promoter expression) or preventing the positioning of Lrp bound at upstream sites for productive interaction with the promoter.
doi:10.1128/JB.184.19.5293-5300.2002
PMCID: PMC135361  PMID: 12218014

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