Introduction

Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA). We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein- (MCP)-1 and evaluated the role of OPN in mediating these effects.

Methods

MCP-1 mRNA and protein in PDA cells treated with or without nicotine (3–300 nM) or OPN (0.15–15 nM) were analyzed by real time PCR and ELISA. Luciferase-labeled promoter studies evaluated the effects of nicotine and OPN on MCP-1 transcription. Intracellular and tissue colocalization of OPN and MCP-1 were examined by immunofluorescence and immunohistochemistry.

Results

Nicotine treatment significantly increased MCP-1 expression in PDA cells. Interestingly, blocking OPN with siRNA or OPN antibody abolished these effects. Transient transfection of the OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P<0.05) increased MCP-1 mRNA and protein and induced its promoter activity. MCP-1 was found in 60% of invasive PDA lesions, of which 66% were smokers. MCP-1 colocalized with OPN in PDA cells and in the malignant ducts, and correlated well with higher expression levels of OPN in the tissue from patients with invasive PDA.

Conclusions

Our data suggest that cigarette smoking and nicotine may contribute to PDA inflammation through inducing MCP-1 and provide a novel insight into a unique role for OPN in mediating these effects.

Osteopontin (OPN) is a pleiotropic protein implicated in various inflammatory responses including ischemia-reperfusion (I-R) injury. Two distinct forms of the protein have been identified: an extensively studied secreted form (sOPN) and a less-well-known intracellular form (iOPN). Studies have shown that increased OPN expression parallels the time course of macrophage infiltration into injured tissue, a late event in the development of cerebral infarcts. sOPN has been suggested to promote remodeling of the extracellular matrix in the brain; the function of iOPN may be to facilitate certain signal transduction processes. Here, we studied OPN expression in adult male Sprague-Dawley rats subjected to global forebrain I-R injury. We found iOPN in the cytoplasm of both cortices and the hippocampus, but unexpectedly only the right cortex exhibited a marked increase in the iOPN level after 45 min of reperfusion. Acetaminophen, a drug recently shown to decrease apoptotic incidence, caspase-9 activation, and mitochondrial dysfunction during global I-R, significantly inhibited the increase in iOPN protein in the right cortex, suggesting a role for iOPN in the response to I-R injury in the right cortex.

Introduction

Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype. We showed recently that nicotine, a major risk factor in pancreatic ductal adenocarcinoma (PDA), increases OPN expression in PDA cells. An OPN splice variant, OPNc, supports anchorage independence and maybe the most potent OPN isoform to convey metastatic behavior. In this study, we tested the effect of nicotine on OPNc expression, and analyzed the correlation between total OPN/OPNc levels and patients’ smoking history.

Methods

Real time PCR and UV-light-illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and in PDA cells treated with or without nicotine (3-300 nM). OPN and OPNc were localized by immunohisotchemistry, and ELISA was used to analyze OPN serum levels.

Results

Nicotine treatment of PDA cells selectively induced denovo expression of OPNc. OPNc was found in 87% of invasive PDA lesions, of which 73% were smokers. The levels of OPNc correlated well with higher expression levels of total OPN in the tissue and serum from patients with invasive PDA.

Conclusions

Our data suggest that smoking and nicotine may contribute to PDA metastatic potential through promoting OPNc expression. Although the direct role of OPNc in PDA progression is not defined, OPNc may have value as a diagnostic and prognostic marker, especially in invasive PDA.

Osteopontin expression has previously been demonstrated in the adult rat dorsal root ganglion, although its function remains unclear. Here, we demonstrate, using real-time reverse transcription-polymerase (RT-PCR) chain reaction, that osteopontin mRNA expression is increased 1 and 3 weeks following sciatic nerve section (axotomy). Further, immunohistochemical staining suggests that this increase is restricted to neurons already expressing the protein. Osteopontin knock-out animals have significantly increased mechanosensory thresholds in the intact adult compared with the wild-type controls; however no differences in allodynia are noted between genotypes using a model of neuropathic pain. Lastly, exogenous recombinant osteopontin has no effect on neurite outgrowth from adult wild-type sensory neurons, nor were differences in neurite outgrowth observed in osteopontin knock-out animals compared with wild-type controls.

Osteopontin (OPN) knockout mice (OPN-KO mice) died of Plasmodium chabaudi chabaudi infection, although wild-type (WT) mice had self-limiting infections. OPN was detected in the WT mice at 2 days postinfection. OPN-KO mice produced significantly smaller amounts of interleukin-12 and gamma interferon than WT mice produced. These results suggested that OPN is involved in Th1-mediated immunity against malaria infection.

Osteopontin (OPN) is a sialated phosphoprotein found in tissues and secreted into body fluids. It is an integrin ligand with pleiotropic functions as an extracellular matrix protein in mineralized tissues and a cytokine that is active in cell signaling (A. B. Tuck, C. Hota, S. M. Wilson, and A. F. Chambers, Oncogene 22:1198-1205, 2003). To determine whether OPN may be important in mucosal defense against viral pathogens, we evaluated the OPN response to rotavirus infection and the extent of diarrhea manifested by infected opn null mutant (opn−/−) mice. Reverse transcription-PCR, Northern and Western blots, and immunohistochemical studies of the HT-29 intestinal epithelial cell line and murine intestine were used to evaluate OPN mRNA and product. Intestinal closed loops and diarrheal observations determined disease severity and duration. OPN mRNA levels increased after infection of HT-29 cells, peaking in 4 to 6 h. Infected cultures contained 925 μg of OPN/ml, while for controls the levels were below detection (50 μg/ml). Infection increased OPN mRNA levels in intestinal tissue between 2 and 24 h postinoculation and increased OPN protein in intestinal fluid. The cellular localization of OPN was supranuclear and apical, and responding cells were diffusely distributed on the villus surface. Three days after infection, closed intestinal loops from opn−/− mice contained more fluid than loops from controls, although secretion levels at the onset of illness were similar. Null mutant mice experienced more intense and prolonged diarrhea than controls. Rotavirus infection of intestinal epithelial cells and murine intestine caused marked increases in OPN mRNA levels and secreted OPN protein. OPN-deficient mice suffered prolonged disease.

Several genetic loci in the mouse have been identified that regulate the severity of Lyme arthritis. The region of chromosome 5 including the osteopontin (OPN) gene (Opn) has been identified in intercross populations of C3H/HeN × C57BL/6 and C3H/HeJ × BALB/cAnN mice. OPN is of particular interest as it is involved in the maintenance and remodeling of tissue during inflammation, it regulates production of interleukin-10 (IL-10) and IL-12 (cytokines implicated in Lyme arthritis), it is necessary for host control of certain bacterial infections, and mice displaying different severities of Lyme arthritis possess different alleles of the OPN gene. Macrophages and splenocytes from OPN-deficient mice on mixed C57BL/6J-129S or inbred 129S backgrounds were stimulated with the Pam3Cys modified lipoprotein from Borrelia burgdorferi, OspA. OPN was not required for OspA-induced cytokine production; however, macrophages from 129S-Opn−/− mice displayed a reduced level of IL-10 production. OPN was also not required for resistance to severe arthritis, as B. burgdorferi-infected 129S-Opn−/− mice developed mild arthritis, as did their wild-type littermates. Arthritis was more severe in OPN-deficient mice on the mixed C57BL/6J-129S backgrounds than in inbred mice of either strain. This increase was most likely due to a gene(s) closely linked to Opn on chromosome 5 in conjunction with other randomly assorting genes. Deficiency in OPN did not influence the numbers of spirochetes in tissues from B. burgdorferi-infected mice, indicating OPN is not part of the host defense to this pathogen. Interestingly, there was no alteration in the B. burgdorferi-specific antibody isotypes in OPN-deficient mice, indicating that its effect on helper T-cell responses is not relevant to the host response to B. burgdorferi.

Reduced mechanical stress to bone in bedridden patients and astronauts leads to bone loss and increase in fracture risk which is one of the major medical and health issues in modern aging society and space medicine. However, no molecule involved in the mechanisms underlying this phenomenon has been identified to date. Osteopontin (OPN) is one of the major noncollagenous proteins in bone matrix, but its function in mediating physical-force effects on bone in vivo has not been known. To investigate the possible requirement for OPN in the transduction of mechanical signaling in bone metabolism in vivo, we examined the effect of unloading on the bones of OPN−/− mice using a tail suspension model. In contrast to the tail suspension–induced bone loss in wild-type mice, OPN−/− mice did not lose bone. Elevation of urinary deoxypyridinoline levels due to unloading was observed in wild-type but not in OPN−/− mice. Analysis of the mechanisms of OPN deficiency–dependent reduction in bone on the cellular basis resulted in two unexpected findings. First, osteoclasts, which were increased by unloading in wild-type mice, were not increased by tail suspension in OPN−/− mice. Second, measures of osteoblastic bone formation, which were decreased in wild-type mice by unloading, were not altered in OPN−/− mice. These observations indicate that the presence of OPN is a prerequisite for the activation of osteoclastic bone resorption and for the reduction in osteoblastic bone formation in unloaded mice. Thus, OPN is a molecule required for the bone loss induced by mechanical stress that regulates the functions of osteoblasts and osteoclasts.

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage φX174 by P1 transduction. φX174 reproduces in this strain at 30 C but not at 40 C. All three stages of φX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of φX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.

The rep gene function of Escherichia coli is essential for the replication of P2 and φX174 double-stranded deoxyribonucleic acid (DNA). Compared with isogenic rep+ strains, rep mutants show the following characteristics: larger cell size, more DNA per cell, and a slightly lower DNA/mass ratio. The replicating rep chromosomes show a steeper gradient of marker frequencies and contain more replicating forks per chromosome. The nucleoid body of rep mutants sediments faster and contains more DNA. We deduce that the rep function is required for the “normal” replication of the E. coli chromosome and that in its absence the E. coli chromosome replicates in an altered manner, perhaps involving slower-moving replicating forks.

Linear φX174 single-stranded DNA can be isolated from φX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5′ end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded φX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.

We have used the single-strand specific nuclease from Neurospora crassa and chromatography on methylated albumin-kieselguhr to purify and characterize repeated and self-complementary sequences from Escherichia coli DNA. Approximately 0.5% of the genome renatures spontaneously at zero time and another 2% renatures somewhat more rapidly than the total DNA. The early renaturing DNA has a base composition and a Tm similar to the total DNA and contains on the average 100 base pairs; the self-complementary DNA also has a base composition like E. coli but contains a mean of 170 base pairs. No evidence was obtained for the presence of a highly redundant sequence.