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Acupuncture increasingly is accepted as a potential therapy for many diseases in the Western world. However, the mechanism of acupuncture is not well understood mechanistically. We have established that manual acupuncture (MA) at the Neiguan (P6) acupoint inhibits excitatory cardiovascular reflex responses through modulation of the autonomic nervous system in the brainstem. It is unclear whether P6 MA activates neurons in the brain regions beyond the brainstem. Thus, we mapped P6 specific neural activation by MA in the forebrain using the Fos-CreER; Ai9 mice model, which allows for enhanced sensitivity and efficiency compared to conventional immunohistochemical staining. Compared to sham-MA control without manual stimulation, we find that MA at P6 markedly increases c-Fos positive neurons in a number of the forebrain regions (n = 5 in each group). These activated regions include accumbens nucleus, caudate putamen, claustrum, bed nucleus of the stria terminalis, amygdaloid nucleus, ventral posterior division of the thalamic nucleus, paraventricular hypothalamic nucleus, arcuate hypothalamic nucleus, primary and secondary somatosensory cortex, ectorhinal cortex, and dorsolateral entorhinal cortex. As MA at P6 activates neurons in relatively broad brain networks beyond the brainstem, our data suggest that acupuncture at this acupoint has the potential to influence physiological functions associated with autonomic and non-autonomic nervous systems through its effects on multiple brain regions., (© 2019 Wiley Periodicals, Inc.)

Cannabinoid receptor-1 (CB1) represents a potential drug target against conditions that include obesity and substance abuse. However, drug trials targeting CB1 (encoded by the CNR1 gene) have been compromised by differences in patient response. Toward addressing the hypothesis that genetic changes within the regulatory regions controlling CNR1 expression contribute to these differences, we characterized the effects of disease-associated allelic variation within a conserved regulatory sequence (ECR1) in CNR1 intron 2 that had previously been shown to modulate cannabinoid response, alcohol intake, and anxiety-like behavior. We used primary cell analysis of reporters carrying different allelic variants of the human ECR1 and found that human-specific C-allele variants of ECR1 (ECR1(C)) drove higher levels of CNR1prom activity in primary hippocampal cells than did the ancestral T-allele and demonstrated a differential response to CB1 agonism. We further demonstrate a role for the AP-1 transcription factor in driving higher ECR1(C) activity and evidence that the ancestral t-allele variant of ECR1 interacted with higher affinity with the insulator binding factor CTCF. The cell-specific approaches used in our study represent an important step in gaining a mechanistic understanding of the roles of noncoding polymorphic variation in disease and in the increasingly important field of cannabinoid pharmacogenetics., (© 2019 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

Destructive bone diseases caused by osteolysis are increasing in incidence. They are characterized by an excessive imbalance of osteoclast formation and activation. During osteolysis, the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are triggered by receptor activator of NF-κB ligand (RANKL), inflammatory factors, and oxidative stress. Previous studies have indicated that the common flavanone glycoside compound hesperetin exhibits anti-inflammatory and antioxidant activity by inhibition of NF-κB and MAPK signaling pathways. However, the direct relationship between hesperetin and osteolysis remain unclear. In the present study, we investigated the effects of hesperetin on lipopolysaccharide (LPS)-induced osteoporosis and elucidated the related mechanisms. Hesperetin effectively suppressed RANKL-induced osteoclastogenesis, osteoclastic bone resorption, and F-actin ring formation in a dose-dependent manner. It also significantly suppressed the expression of osteoclast-specific markers including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1. Furthermore, it inhibited osteoclastogenesis by inhibiting activation of NF-κB and MAPK signaling, scavenging reactive oxygen species, and activating the nuclear factor E2 p45-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway. Consistent with in vitro results, hesperetin effectively ameliorated LPS-induced bone loss, reduced osteoclast numbers, and decreased the RANKL/OPG ratio in vivo. As such, our results suggest that hesperetin may be a great candidate for developing a novel drug for destructive bone diseases such as periodontal disease, tumor bone metastasis, rheumatoid arthritis, and osteoporosis., (© 2018 Wiley Periodicals, Inc.)

Skeletal homeostasis relies upon a fine tuning of osteoclast (OCL)-mediated bone resorption and osteoblast (OBL)-dependent bone formation. This balance is unsettled by multiple myeloma (MM) cells, which impair OBL function and stimulate OCLs to generate lytic lesions. Emerging experimental evidence is disclosing a key regulatory role of microRNAs (miRNAs) in the regulation of bone homeostasis suggesting the miRNA network as potential novel target for the treatment of MM-related bone disease (BD). Here, we report that miR-29b expression decreases progressively during human OCL differentiation in vitro. We found that lentiviral transduction of miR-29b into OCLs, even in the presence of MM cells, significantly impairs tartrate acid phosphatase (TRAcP) expression, lacunae generation, and collagen degradation, which are relevant hallmarks of OCL activity. Accordingly, expression of cathepsin K and metalloproteinase 9 (MMP9) as well as actin ring rearrangement were impaired in the presence of miR-29b. Moreover, we found that canonical targets C-FOS and metalloproteinase 2 are suppressed by constitutive miR-29b expression which also downregulated the master OCL transcription factor, NAFTc-1. Overall, these data indicate that enforced expression of miR-29b impairs OCL differentiation and overcomes OCL activation triggered by MM cells, providing a rationale for miR-29b-based treatment of MM-related BD., (Copyright © 2012 Wiley Periodicals, Inc.)

Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N-Ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-Ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior., (Copyright © 2012 Wiley Periodicals, Inc.)

Human proto-oncogene c-Jun and c-Fos assemble the activator protein-1 complex which is a crucial transcription factor responding to environmental factors and promotes tumorgenesis. We hypothesized that genetic variants in these two genes may alter the carriers' susceptibility to lung cancer. In two independent case-control studies, we genotyped three putative functional polymorphisms (-1318T>G and -673T>C of c-Jun; -60C>T of c-Fos) in southern Chinese and then validated the association in eastern Chinese. We found that compared to -1318TT genotype, the -1318GT/GG variant genotypes had an increased lung cancer risk (OR=1.46, 95% CI=1.26-1.69), and the -673CC genotype had an increased lung cancer risk compared to -673TT/CT genotypes (OR=1.35, 95% CI=1.17-1.56) in the total 1,559 cases versus 1,679 controls. After combining these two loci, the number of the risk genotypes was associated with increased cancer risk in a dose-response manner (ptrend=2.21×10(-11)); moreover, the risk genotypes interacted with smoking or drinking status on increasing cancer risk (p values of interaction were 0.009 and 0.007, respectively). Further, we found that those with -1318GT/GG genotypes, -673CC genotypes or both genotypes in c-Jun had higher mRNA and protein expression levels in vivo, and those variants had higher transcription activities in reporter genes in vitro, especially under the stimuli with tobacco extract or alcohol mixture as luciferase assay shown. However, for -60C>T of c-Fos, no significant association was observed for lung cancer risk. Our data suggested that the genetic variants in c-Jun (-1318T>G and -673T>C) increase the carriers' susceptibility to lung cancer via interaction with smoking or drinking on increasing the c-Jun's expression., (Copyright © 2011 UICC.)

Transcription factor activator protein-1 (AP-1) super-family is known to modulate expression of array of genes during development of many cancers and considered as an important target for modern therapeutics. But the role of AP-1 during development of human oral cancers is still poorly understood. Because oral cancer is one of the most common cancers in India and south-east Asia, we studied the activation and expression pattern of AP-1 family of proteins and mRNA in different stages of oral carcinogenesis. Gel-shift assay, western blotting, immunohistochemistry and northern blotting have been used to assess the binding activity and expression pattern of AP-1 family (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) proteins and mRNA transcripts in a total of 100 fresh oral tissue specimens comprising precancer (n = 40), cancer (n = 50) and healthy control (n = 10). Constitutive activation of AP-1 with concomitant upregulated expression of majority of AP-1 family of proteins and mRNA was observed in cancer cases. Interestingly, almost all precancerous cases showed JunD homodimers, whereas c-Fos/JunD was the most prevalent complex found in cancer tissues. The overexpression of EGFR mRNA, p50:p50/NF-kappaB homodimer formation, together with overexpression of pERK and c-Fos proteins in this study suggests an interesting cross talk between AP-1 and NF-kappaB pathways in oral cancers. Thus, this study demonstrates differential expression and activation of AP-1 super-family proteins in relation to severity of lesion and their crucial role in human oral carcinogenesis.

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK., ((c) 2007 Wiley-Liss, Inc.)

Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron 1. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron 1 is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes., ((c) 2006 Wiley-Liss, Inc.)

Perinatal exposure to diethylstilbestrol (DES) induces reproductive tract cancers later in life in both humans and animals. Because there is no clear evidence that perinatal DES exposure induces gene mutation, we proposed that perinatal DES exposure causes epigenetic methylation changes that result in persistent alterations in gene expression, leading to tumorigenesis. The proto-oncogene c-fos is one of the immediately induced genes in uterine epithelium after estrogen simulation and a key player in uterine carcinogenesis. Here, we investigated c-fos expression in mice neonatally exposed to DES (2 microg/pup/day on postnatal days 1-5). The mRNA levels of c-fos in uteri of neonatal DES-treated mice were persistently 1.4-1.9-fold higher than that in the control mice from day 5 to day 60. Overall, the uterine c-fos expression level in the neonatal DES-exposed group was significantly higher than that in the control group. After examination of the methylation status of the c-fos gene, we found that the CpGs in promoter and intron-1 regions were completely unmethylated. In exon-4, from day 17 to day 60, the percentage of unmethylated CpGs was higher in neonatal DES-exposed mice uteri than that in control (42%, 51%, 47%, and 42% in DES-exposed mice vs 33%, 34%, 33%, and 21% in control mice at day 17, 21, 30, and 60, respectively). These results suggest that perinatal DES exposure may permanently alter gene expression and methylation, and the methylation modification may occur in either the promoter regions or other regulatory sites in the gene., (Published 2003 Wiley-Liss, Inc.)

The central nucleus of the amygdala (CeA) is activated robustly by an immune challenge such as the systemic administration of the proinflammatory cytokine interleukin-1beta (IL-1beta). Because IL-1beta is not believed to cross the blood-brain barrier in any significant amount, it is likely that IL-1beta elicits CeA cell recruitment by means of activation of afferents to the CeA. However, although many studies have investigated the origins of afferent inputs to the CeA, we do not know which of these also respond to IL-1beta. Therefore, to identify candidate neurons responsible for the recruitment of CeA cells by an immune challenge, we iontophoretically deposited a retrograde tracer, cholera toxin b-subunit (CTb), into the CeA of rats 7 days before systemic delivery of IL-1beta (1 microg/kg, i.a.). By using combined immunohistochemistry, we then quantified the number of Fos-positive CTb cells in six major regions known to innervate the CeA. These included the medial prefrontal cortex, paraventricular thalamus (PVT), ventral tegmental area, parabrachial nucleus (PB), nucleus tractus solitarius, and ventrolateral medulla. Our results show that after deposit of CTb into the CeA, the majority of double-labeled cells were located in the PB and the PVT, suggesting that CeA cell activation by systemic IL-1beta is likely to arise predominantly from cell bodies located in these regions. These findings may have significant implications in determining the central pathways involved in generating acute central responses to a systemic immune challenge., (Copyright 2002 Wiley-Liss, Inc.)

Nordihydroguaiaretic acid (NDGA) is a polyphenolic compound from the Larrea tridentata bush that has been identified as a chemopreventive drug in animal studies. Topically applied NDGA has been shown to prevent phorbol ester promotion of tumors in mouse skin, suggesting that NDGA may be a candidate drug for the chemoprevention of skin cancer. Ultraviolet (UV) B irradiation from sunlight exposure is the major cause of human skin cancer. UVB irradiation causes epigenetic alterations in target keratinocytes, such as the upregulation of signal transduction pathways that induce the expression of transcription factors. Specifically, UVB induces activator protein-1 (AP-1), a transcription factor complex that alters normal cellular gene expression. A component of the UVB-induced AP-1 complex, c-fos, also was identified as a mediator of the signaling pathway that leads to AP-1 activation. Thus, NDGA was investigated as a potential inhibitory agent for UVB-induced signaling pathways in the human keratinocyte cell line HaCaT. NDGA significantly inhibited UVB-induced c-fos and AP-1 transactivation. In addition, NDGA was found to inhibit activity of phosphatidylinositol 3-kinase (PI 3-kinase), a UVB-inducible enzyme that contributes to the induced expression of c-fos and AP-1. Therefore, NDGA prevents UVB-induced c-fos expression and AP-1 transactivation by inhibiting the PI 3-kinase signaling pathway. Effective skin chemoprevention strategies may incorporate NDGA to inhibit components of the UVB-induced cell signaling pathways that increase AP-1 activity., (Copyright 2002 Wiley-Liss, Inc.)

To characterize the temporal expression of genes that play a functional role during the process of osteoblast adhesion, we used differential display (DD-PCR) on mRNA isolated from attached vs. suspended osteoblasts. A 200-bp fragment displaying upregulated expression after 30 and 60 min adhesion was isolated, sequenced, and showed 97% homology to prtb, previously showed to be expressed in mouse brain. Northern analysis confirmed a two-fold increase in prtb message during adhesion to tissue culture polystyrene, both in the presence or absence of surface-adsorbed serum proteins. Serum stimulation alone was also able to induce prtb expression, although to a lesser extent, in suspension cells. Strong prtb expression was also detected in both brain and bone of adult rats. Furthermore, prtb expression analysis during MC3T3-E1 cell differentiation revealed high expression levels independent of proliferation (day 0-7), matrix maturation (day 7-14), and mineralization (day 14-31). Time course analysis of prtb expression during adhesion of sensitized osteoblasts to serum-protein coated surfaces showed robust mRNA expression at 5 min post-plating and a peak at 10 min. The two known serum-inducible immediate early genes c-fos and c-jun showed similar expression kinetics, with c-jun mRNA levels peaking at 15 min and c-fos at 20 min. Based on these data, we hypothesize that prtb may function as an immediate early, serum-responsive, and adhesion-inducible gene with possible involvement in processes such as cell cycle control, adhesion, and proliferation., (Copyright 2001 Wiley-Liss, Inc.)

Adrenomedullin (ADM), encoded by the preproadrenomedullin (ppADM) gene, exerts multiple effects in a wide variety of peripheral and central tissues. Although ADM-like immunoreactivity has been shown to be widely distributed throughout the rat central nervous system (CNS), the detailed distribution of ppADM gene expression in the CNS and its modulation by physiological stimuli remain unknown. In our study, in situ hybridization was used to localize ppADM mRNA in the rat brain and to quantify its levels after exposure to different stressors including lipopolysaccharide (LPS; 100 microg/kg, iv), restraint stress (2 cycles of 1 hour restraint/1 hour rest), and 24 hours of dehydration. In addition, Fos immunoreactivity was used to identify the activation of neurons in response to LPS. Our results show that ppADM mRNA is widely distributed throughout the rat CNS, with especially high levels in autonomic centers including the hypothalamic paraventricular nucleus (PVN), hypothalamic supraoptic nucleus (SON), locus coeruleus, ventrolateral medulla, and intermediolateral cell column of the spinal cord. Furthermore, LPS inhibits ppADM gene expression in the parvocellular PVN (pPVN), magnocellular PVN (mPVN), SON, dorsal motor nucleus of the vagus, and area postrema among examined regions; restraint stress reduces ppADM mRNA levels in the pPVN, mPVN, SON, nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, and subfornical organ; 24 hours of water deprivation decreases ppADM gene expression only in the mPVN and SON. Taken together, our results suggest that ADM is involved in the regulation of the hypothalamo-neurohypophysial system, the hypothalamo-pituitary-adrenal axis, and central autonomic functions., (Copyright 2001 Wiley-Liss, Inc.)

Background: Ciliary neurotrophic factor (CNTF) is a member of the interleukin-6 (IL-6) cytokine family and affects the survival and differentiation of several classes of neurons. For signal transduction, CNTF requires a receptor complex, composed of the IL-6 signal transducing molecule gp130, leukemia inhibitory factor receptor (LIFR)-beta, and CNTFR-alpha. There are two major independent pathways (Jak-STAT and Ras-MAPK) in cell signaling, and some recent reports show interaction between these pathways. The signal of the IL-6 family is mainly transduced to the Jak-STAT pathway through gp130. However, it has not been examined in neuroblastoma in detail., Procedure and Results: Here we examine the signaling pathway of CNTF in 11 neuroblastoma cell lines. Northern blot analysis revealed that 3 of the 11 cell lines expressed c-fos mRNA after CNTF stimulation. Cell lysates were immunoprecipitated with agarose-conjugated antiphosphotyrosine antibody and blotted with anti-gp130, anti-Jak1, or anti-STAT3 antibody. Tyrosine phosphorylation of gp130, Jak1, and STAT3 was observed after CNTF stimulation in these three cell lines. Furthermore, tyrosine phosphorylation of ERK1 (one of the MAPKs) was also observed in all of them., Conclusions: These results demonstrate that CNTF signaling is conserved in some of the neuroblastoma cell lines and suggest that not only a Jak-STAT pathway but a MAPK pathway is activated by CNTF through gp130 in neuroblastoma cell lines.

SV40 large T antigen-induced primitive neuroectodermal tumors of the rat provide a model system to study induction and progression of primitive neuroectodermal tumors at the molecular level. A cell line derived from such a tumor reproducibly gave rise to malignant derivatives that ceased large T-antigen expression but harbored a mutant p53 allele with a common mutation at Cys(174) to Tyr (C174Y). To determine whether this p53 mutation contributes to tumor progression, we analyzed mutant C174Y functionally. Co-transfection experiments in Saos-2 cells with mutant or wild-type p53 and reporter genes linked to various p53-responsive promoters revealed that mutant C174Y failed to transcriptionally transactivate the Mdm2, Waf1, Cyclin G and Bax promoters. Loss of transcriptional activation correlated with loss of DNA-binding activity. Moreover, mutant C174Y exhibited a dominant negative effect on co-expressed wild-type p53. The ability of mutant p53 to repress the viral RSV, LTR or SV40 early promoters or the cellular fos promoter was likewise impaired. In contrast, it showed even induction of the fos promoter. Consistent with these observations, mutant C174Y was non-functional in the suppression of Saos-2 cell growth and even conferred a growth advantage to the cells. Surprisingly, mutant C174Y was also impaired in nuclear transport, as revealed by immunofluorescence analyses. Taken together, our results demonstrate that mutant C174Y possesses features that can positively contribute to cancer progression., (Copyright 2000 Wiley-Liss, Inc.)

The effect of binocular central retinal lesions on the expression of the immediate early genes c-fos and zif268 in the dorsal lateral geniculate nucleus (dLGN) and the visual cortex of adult cats was investigated by in situ hybridization and immunocytochemistry. In the deafferented region of the dLGN, the c-fos mRNA level was decreased within 3 days. The dimensions of the geniculate region showing decreased amounts of c-fos mRNA matched the predictions based on the lesion size and the retinotopic maps of Sanderson ([1971] J. Comp. Neurol. 143:101-118). We did not detect zif268 mRNA in the dLGN. At the cortical level, both c-fos and zif268 mRNA expression decreased in the sensory-deprived region of area 17. In addition, the portions of areas 18, 19, 21a, 21b, and 7, as well as the posterior medial lateral suprasylvian area, the posterior lateral lateral suprasylvian area, the ventral lateral suprasylvian area, and the dorsal lateral suprasylvian area corresponding to the retinal lesions also displayed decreased c-fos and zif268 mRNA levels. Immunocytochemistry revealed similar changes for Zif268 and Fos protein. Three days post lesion, the dimensions of the lesion-affected cortical loci exceeded the predictions in relation to the size of the retinal lesions and the available retinotopic maps. Longer postlesion survival times clearly resulted in a time-dependent restoration of immediate early gene expression from the border to the center of the lesion-affected cortical portions. Our findings represent a new approach for investigating the capacity of adult sensory systems to undergo plastic changes following sensory deprivation and for defining the topographic nature of sensory subcortical and cortical structures., (Copyright 2000 Wiley-Liss, Inc.)

The EPC-1 (early population doubling level cDNA-1) gene, also known as pigment epithelium-derived factor, encodes a protein belonging to the serine protease inhibitor (serpin) superfamily that has been reported to inhibit angiogenesis and proliferation of several cell types. We have previously reported that the EPC-1 mRNA and the secreted EPC-1 protein are expressed at levels more than 100-fold higher in early passage, G(0), WI-38 cells compared to either proliferating or senescent WI-38 fibroblasts. To examine the molecular mechanisms that regulate changes in EPC-1 gene expression in WI-38 cells, we isolated and characterized the human EPC-1 gene and determined the mRNA cap site. Transcriptional assays showed no change in the transcription rates of EPC-1 between young proliferating, quiescent, and senescent WI-38 cells. These results suggest posttranscriptional regulation of the EPC-1 gene. Reverse transcriptase polymerase chain reaction measurements (of hnRNA) indicate regulation at the hnRNA level. The regulation of the EPC-1 gene at the level of hnRNA can explain the observed slow increase in the steady-state EPC-1 mRNA levels when cells become quiescent. The reduction of EPC-1 mRNA levels that occurs when cells exit G(0) and are induced to proliferate can be accounted for by a reduction of the EPC-1 mRNA stability in stimulated cells as compared to quiescent cells., (Copyright 2000 Wiley-Liss, Inc.)

Previous studies in this laboratory have shown that the SH-2 domain-containing protein tyrosine phosphatase SHP-1 is expressed in CNS glia and functions to modulate cytokine activities in these cells. The present study demonstrates that SHP-1 is expressed within multiple regions of the CNS in vivo, especially in white matter. Interestingly, we show that mice genetically lacking in SHP-1 (motheaten mice) in the CNS displayed dysmyelination. We therefore examined the expression of SHP-1 in the myelin-forming oligodendrocytes. Oligodendrocytes present in either mixed glial cultures or pure cultures expressed high levels of SHP-1 in the cytoplasm of cell bodies and processes. Oligodendrocytes isolated from motheaten mice did not express SHP-1. To test possible functions for SHP-1 in oligodendrocytes in controlling cytokine signaling, we compared the responsiveness of oligodendrocytes isolated from either motheaten or normal littermate mice with IL-6. IL-6 induced higher levels of STAT3 phosphorylation and STAT3-responsive c-fos gene expression in pure oligodendrocyte cultures of motheaten compared with normal littermate mice. These studies demonstrate that oligodendrocytes express SHP-1 and that SHP-1 functions to control IL-6 signaling. SHP-1 may therefore be a critical regulator of oligodendrocyte differentiation in response to IL-6 family cytokines. Further, these findings may relate to dysmyelination in mice lacking SHP-1., (Copyright 2000 Wiley-Liss, Inc.)

In this report, the authors provide a novel description of a population of gamma-aminobutyric acid-containing neurons in the substantia nigra, pars compacta (SNC). By using metabolic mapping of the immediate-early gene, c-fos, the activation pattern of these cells was characterized with respect to basal ganglia stimulation. Dopaminergic stimulation with d-amphetamine or apomorphine induced Fos expression in the central region of the SNC. However, lesions of the nigrostriatal dopamine pathway significantly reduced d-amphetamine- and apomorphine-induced Fos expression in the ipsilateral and contralateral SNC, respectively. Suppression of stimulant-induced Fos expression in the striatum, using antisense oligodeoxynucleotides, also eliminated Fos expression in the ipsilateral SNC, indicating that striatal efferent projections are involved in the activation of these cells. Double-labeling immunohistochemistry revealed that the Fos-positive cells did not express tyrosine hydroxylase but were immunoreactive for glutamic acid decarboxylase. Retrograde labeling of nigrostriatal neurons, combined with Fos immunofluorescence, revealed that these Fos-positive cells did not project to the striatum. Thus, these neurons do not appear to comprise a nondopaminergic nigrostriatal circuit but likely represent locally-projecting interneurons of the substantia nigra., (Copyright 2000 Wiley-Liss, Inc.)

To study oncoprotein cooperation in vivo, transgenic mice were established that coexpressed human transforming growth factor-alpha (TGFalpha) and v-fos exclusively in the epidermis by means of a human keratin 1 (HK1)-based vector. HK1.fos/alpha mice exhibited aberrant epidermal proliferation and differentiation and formed spontaneous papillomas that achieved tumor autonomy but did not convert to malignancy. To determine the sensitivity to a chemical promotion stimulus, HK1.fos/alpha mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Previously, after 7 mo TPA promotion of HK1.TGFalpha mice that express moderate levels of TGFalpha elicited papillomas that remained regression-prone and benign for up to 2 yr. In HK1.fos mice, 6 mo TPA elicited papillomas that required spontaneous c-Ha-ras activation and converted to malignancy after 14-16 mo. We now show that in HK1.fos/alpha transgenic genotypes, TPA promotion accelerated papillomatogenesis, with the earliest papilloma appearance at 2 mo after initiation of TPA promotion. These papillomas started to convert to malignancy by 10 mo. Analysis of HK1.fos/alpha papillomas and carcinomas revealed that the endogenous c-Ha-ras gene possessed mutations at codons 12, 13, and 61 at the papilloma stage, but no mutations of the p53 tumor suppressor gene were detected. These data indicate that coexpression of fos and TGFalpha increased epidermal sensitivity to TPA promotion, which accelerated malignant conversion. However, in this transgenic model conversion always required additional genetic events, e.g., activation of the endogenous c-Ha-ras gene., (Copyright 1999 Wiley-Liss, Inc.)

This study was designed to systematically examine the effects of persistent orofacial tissue injury on prolonged neuronal activation in the trigeminal nociceptive pathways by directly comparing the effects of orofacial deep vs. cutaneous tissue inflammation on brainstem Fos protein expression, a marker of neuronal activation. Complete Freund's adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. Rats were perfused 2 hours, 24 hours, 3 days, or 10 days following CFA injection. The TMJ and PO inflammation-induced Fos expression paralleled the intensity and course of inflammation over the 10-day observation period, suggesting that the increase in intensities and persistence of Fos protein expression may be associated with a maintained increase in peripheral input. Compared to PO CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a greater Fos expression. In TMJ- but not in PO-inflamed rats, Fos-like immunoreactivity (LI) spread from superficial to deep upper cervical dorsal horn as the inflammation persisted and there was a dominant ipsilateral Fos-labeling in the paratrigeminal nucleus. Common to TMJ and PO inflammation, Fos-LI was induced in the trigeminal subnuclei interpolaris and caudalis, C1-2 dorsal horn, and other medullary nuclei. Substantial bilateral Fos-LI was found in the interpolaris-caudalis trigeminal transition zone. Further analysis revealed that Fos-LI in the ventral transition zone was equivalent bilaterally, whereas Fos-LI in the dorsal transition zone was predominantly ipsilateral to the inflammation. The differential induction of Fos expression suggests that an increase in TMJ C-fiber input after inflammation and robust central neuronal hyperexcitability contribute to persistent pain associated with temporomandibular disorders., (Copyright 1999 Wiley-Liss, Inc.)

This work was designed to test whether the suppression of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinomas by human chorionic gonadotropin (hCG) is associated with the synthesis of inhibin. For this purpose, virgin rats received 8 mg DMBA/100 g body weight; 20 d later they were injected daily with 100 IU of hCG for 40 d (DMBA+hCG group). Age-matched untreated (control), hCG-, and DMBA+saline-treated rats were used for comparisons. Mammary tissues were collected for histopathological and mRNA analyses after 5, 10, 20, and 40 d of hCG injection and 20 d after treatment. None of the animals in the control and hCG-treated groups developed mammary tumors. DMBA-treated rats developed a high incidence of both microscopic lesions, i.e., intraductal proliferations and ductal carcinomas in situ, and palpable tumors. In DMBA+hCG-treated rats, the incidence of microscopic and palpable tumors was markedly reduced. In these animals, alpha- and beta-inhibin immunoreactivity was elevated in the non-tumoral mammary glands in association with lobule formation and in the tumors. Inhibin A and B mRNAs were also elevated in the mammary tissue, and c-myc and c-jun were induced by the hormonal treatment. DMBA alone did not modify the expression of these genes. Our findings indicate that inhibin production and gene activation are associated with both mammary gland differentiation and tumor regression.

We used immunocytochemistry to determine the regional and temporal distribution of Fos protein expression in awake and unrestrained rats after a unilateral stereotaxic microinjection of a cholinergic agonist, carbachol, in the thalamic ventroposterolateral and reticular nuclei, previously shown to cause limbic and generalized convulsive seizures. The microinjection of carbachol elicits behavioral alterations including immobilization, staring, facial and jaw clonus, rearing, and falling, followed by recurrent generalized convulsive seizures, and a pattern of c-fos expression throughout the brain. In addition to the hypothalamic paraventricular and supraoptic nuclei, the initial induction of c-fos expression was observed as early as 15 minutes after the carbachol microinjection, in the piriform and entorhinal cortices, the thalamic paraventricular, the supramammilary, the lateral parabrachial nuclei, and the central gray. From 30 minutes to 2 hours, corresponding to the occurrence of motor expression of limbic and recurrent generalized convulsive seizures, Fos immunoreactivity was seen in a number of functionally related brain regions including the hippocampus, the amygdala, and the anterior thalamic nucleus (limbic system); the thalamus, the basal ganglia, and the cortex (thalamo-striatal-cortical system); and the hypothalamus, the central nucleus of the amygdala, the pons, and the medulla (central autonomic system). On the basis of the present results showing regional and temporal c-fos expression and well known neuroanatomical connections, we have constructed a neural network relating the limbic, thalamo-striatal-cortical, and central autonomic systems. This analysis provides, for the first time, neuronal circuits and pathways relating epilepsy-elicited behavioral expression of convulsive seizures and adaptive homeostatic responses and could serve as a basis for studying central autonomic regulation during epileptic disorders., (Copyright 1999 Wiley-Liss, Inc.)

Bilateral lesions of the central tegmental field (CTF) in male rats virtually eliminate mating behavior. This study examined if mating-induced Fos expression (a measure of neuronal activation) and androgen receptors (AR) are colocalized in brain and spinal cord neurons which project to the CTF. Animals received unilateral injections of the retrograde tracer Fluorogold (FG) in the lateral part of the CTF (CTFl), and 10 days later were killed after ejaculating with females. Brains and spinal cords were examined for FG transport, AR-immunoreactivity (AR-ir), and Fos-immunoreactivity (Fos-ir). AR-ir and Fos-ir were visualized with fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. The CTFl received projections from AR-containing neurons in forebrain structures (bed nucleus of stria terminalis, medial preoptic area, lateral and ventromedial hypothalamus), in the central amygdala and various mid- and hindbrain structures (dorsolateral tegmentum, superior and inferior colliculi, pedunculopontine nucleus), and in the lumbosacral spinal cord (lamina X). Some of the AR-containing neurons in bed nucleus of stria terminalis and in the dorsal part of the medial preoptic area with projections to the CTFl were activated by mating. Most AR-containing neurons in spinal lamina X with projections to the CTFl were also activated by mating. Information from spinal cord and pontine nuclei and from outputs descending from the forebrain may be relayed in the CTFl. Thus, as part of a network of hormone-sensitive neurons linking brain and spinal cord mechanisms for mating, the CTFl could participate in the integration of visceral and somatic information relevant for sexual behavior.

The role of serine/threonine protein phosphatase type 2A (PP2A) in cellular growth control has not yet been thoroughly established. Earlier experiments with okadaic acid, a phosphatase inhibitor, suggested that PP2A may act as an anti-oncogene, although a direct role for this enzyme in the transformation process has not been demonstrated. We therefore investigated whether altered levels of PP2A expression would affect the transformation of mouse fibroblasts by the Ha-ras oncogene. Here we report that cells with elevated levels of PP2A expression were more resistant to focus formation induced by Ha-ras. At the molecular level, this was paralleled by the reduced Ha-ras-stimulated expression of the c-fos promoter, a proto-oncogene target for Ha-ras signaling. Thus, our results support a negative role for PP2A in the process of cellular transformation and may ascribe tumor-suppressing functions to this enzyme.

Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in culture before reaching the end of their proliferative life span, an event termed in vitro cellular senescence. Considerable evidence suggests that altered expression of key genes involved in the mitogenic response may be responsible for the inability of senescent cells to proliferate. Here we examined the expression and activity of the early growth response-1 (egr-1) gene, an "immediate-early" gene that is believed to link extracellular mitogenic signals to cell-cycle progression. We found that egr-1 was strongly downregulated in senescent HDFs at the level of mRNA, protein, and DNA binding activity. Decreased DNA binding activity of Egr-1 in vitro corresponded to decreased transcriptional activation in vivo. To further understand the mechanism of egr-1 downregulation, we examined the potential role of the serum response elements (SREs) present in the egr-1 promoter. Electrophoretic mobility shift studies using young and old cell nuclear extracts showed a marked decrease in serum response factor (SRF) binding activity to the SRE in old compared to young cells. Loss of SRF binding activity has been correlated with the loss of expression of another growth-related immediate-early gene (c-fos). These results suggest a common mechanism for the downregulation of c-fos, egr-1, and other SRE-dependent, mitogen-responsive genes during cellular senescence.

The conversion of a palindromic sequence, GGTCTnnnAGACC, in the 5'-flanking region of the murine c-fos proto-oncogene into a functional estrogen-response element by a single base change into GGTC(A/G)nnnAGACC has previously been postulated [Nawaz et al., 1993] as a possible mechanism of the induction of tumors by estrogens. This attractive hypothesis has been investigated in estradiol-induced Syrian hamster kidneytumors, in H-301 kidney tumor cells (a cell line derived from the Syrian hamster tumor), and in normal kidney tissue. The c-fos gene is differentially regulated by a classical estrogen receptor-mediated process in tumors, whereas in the acutely treated kidney, estradiol induces c-fos expression independent of estrogen-receptor function. In this study, we identified in the 5'-flanking region of the hamster kidney c-fos gene the sequence AGTCCnnnAGACC, which closely resembled but did not appear to function as an estrogen-response element. No mutations were detected in this sequence or in the 5'-flanking region of c-fos genes from three different primary tumors and from H-301 tumor cells. To rule out the possibility of a low copy number of mutant alleles in a tumor sample, polymerase chain reaction-based single-strand conformation polymorphism analysis was performed on 372 base pairs of the 5'-flank of the c-fos gene (-367 to +5 base pairs relative to the transcription start point). Nine different kidney tumor DNA samples and five normal kidney tissue samples (controls) produced an identical pattern of DNA bands, suggesting a lack of natural polymorphisms and mutations in this region of the c-fos gene. Acute treatment of hamsters with 17beta-estradiol for 6 h significantly induced renal c-fos mRNA expression, whereas control levels of c-fos were restored by co-treatment with estradiol and either N-acetyl-L-cysteine or alpha-naphthoflavone. We concluded that the previously observed change in regulatory control of c-fos expression in kidney versus estradiol-induced tumors does not involve the creation of a functional estrogen-response element by single point mutation in the 5'-flanking region of the gene. Additionally, c-fos expression in estradiol-treated hamster kidneys appears to be mediated by free radicals generated by the catechol metabolites of estradiol and not by the activation of any estrogen receptor.

Pulmonary surfactant is a mixture of phospholipids and surfactant-associated proteins made by alveolar type II cells that is necessary for normal lung function. Surfactant secretion and reuptake by type II cells are regulated in part by interaction of surfactant protein-A (SP-A) with a specific receptor (SPAR) on type 11 cells. Several chemicals and hormones affect both surfactant secretion and also surfactant gene expression, but consequences of SP-A-SPAR interaction beyond regulating surfactant secretion and reuptake are unknown. Accordingly, we studied the effects of SP-A on surfactant protein gene transcription, mRNA levels, and transcript stability. SP-A elicited new transcription of surfactant proteins SP-A, SP-B, and SP-C and SPAR and c-Jun but had no effect on beta-actin or c-fos transcription. Antibody against SP-A receptor blocked SP-A-induced transcription, confirming that these actions of SP-A were receptor-mediated. SP-A effects on overall transcript levels were more complex. However, SP-A, SP-B, and SP-C mRNA levels doubled in SP-A-treated cells compared to controls. SP-A is known to stabilize surfactant, control its secretion and reuptake by type II cells, and augment host antimicrobial defenses. These data indicate that SP-A also acts as an autocrine cytokine: it binds its receptor and specifically regulates transcription of surfactant proteins and other genes.

Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).

Bone cells are a prime target for the biological function of the fos/jun (activating protein-1 (AP-1)) transcription factor complex. Deregulated expression of c-fos or v-fos in bone cells induces tumorigenicity and the formation of non-metastatic osteosarcomas. In contrast, fos oncogenes transform fibroblasts to an invasive phenotype accompanied by the expression of various invasion- and metastasis-associated genes. Here we compared the expression of AP-1-dependent genes and AP-1 activity in cell lines from fos-induced, radiation-induced, and spontaneous osteosarcomas. We showed that the presence of high AP-1 activity was not sufficient for the induction of invasion- and metastasis-associated AP-1-dependent genes in transformed bone cells. Further, we identified the collagenase I and stromelysin 1 gene promoters as suitable tools for the analysis of other factors regulating metastatic progression of osteosarcoma.

We have analyzed the ontogenic initiation and maintenance of methylation of certain Hpall (m), Hhal (H), Hincll (Hc), and Sall (SI)-specific CpG sites in the coding region of the proto-oncogene, c-fos, through testicular cells, sperm, and fetal, neonatal, and adult somatic tissues. The results show that 1) sperm-derived methylated sites get demethylated in early development. However, unlike other studied genes, they remain so at least up to day 13.5 post coitum (pc); 2) de novo methylation proceeds unidirectionally in a step-wise, site-specific manner between m5-m3 sites; 3) the mature, tissue-specific, adult methylation pattern is established between day 0 and day 20 of neonatal development; 4) the Hc and SI sites (CGTCGAC), occurring at an interval of one nucleotide, are only partially methylated in all the tissues; and 5) m3 and H1 sites, which occur close to an Sp1 motif, escape methylation in most of the tissues. The present study on the embryonic gene, c-fos, thus provides a novel pattern of de novo methylation in development. Also, it suggests that close proximity of CpGs may prevent methylation.

In this study, Fos immunohistochemistry was used to map brainstem neuronal pathways activated during hypercapnia and hypoxia. Conscious rats were exposed to six different gas mixtures: (a) air; (b) 8% CO2 in air; (c) 10% CO2 in air; (d) 15% CO2 in air; (e) 15% CO2 + 60% O2, balance N2; (f) 9% O2, balance N2. Double-staining was performed to show the presence of tyrosine hydroxylase. Hypercapnia, in a dose-dependent way caused Fos expression in the following areas: caudal nucleus tractus solitarius (NTS), with few labeled A2 noradrenergic neurons; noradrenergic A1 cells and noncatecholaminergic neurons in the caudal ventrolateral medulla; raphe magnus and gigantocellular nucleus pars alpha (GiA); many noncatecholaminergic (and relatively few C1) neurons in the lateral paragigantocellular nucleus (PGCl), and in the retrotrapezoid nucleus (RTN); locus coeruleus (LC), external lateral parabrachial and Kölliker-Fuse nuclei, and A5 noradrenergic neurons at pontine level; and in caudal mesencephalon, the ventrolateral column of the periaqueductal gray (vlPAG). In most of these nuclei, hypoxia also induced Fos expression, albeit generally less than after hypercapnia. However, hypoxia did not cause labeling in RTN, juxtafacial PGCl, GiA, LC, or vlPAG. After normoxic hypercapnia, more labeled cells were present in NTS and PGCl than after hyperoxic hypercapnia. Part of the observed labeling could be caused by stress- or cardiovascular-related sequelae of hypoxia and hypercapnia. Possible implications for the neural control of breathing are also discussed, particularly with regard to the finding that several nuclei, not belonging to the classical brainstem respiratory centres, contained labeled cells.

Increased bcl-2 expression is a common feature of many types of human malignancies, which implies that bcl-2 plays an important role in tumorigenesis. To better understand the molecular mechanisms of bcl-2-induced oncogenesis, we examined the effects of bcl-2 expression on transformation of mouse epidermal JB6 cells induced by the tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA). Promotion-sensitive JB6 clone41 cells were transfected with the bcl-2-containing expression vector pD5-neo/bcl-2, and the soft agar growth of bcl-2-transfected cells and control cells were compared. bcl-2 overexpression in JB6 clone41 cells caused a TPA-induced soft-agar growth fivefold greater than the growth of nontransfected or vector-transfected (neo control) cells. bcl-2 expression in the absence of TPA did not lead to colony formation in soft agar. Because the level of the transcription factor activator protein 1 (AP-1) has been shown to be critical for the responsiveness of JB6 cells to TPA-induced transformation, we compared c-jun and c-fos expression as well as the AP-1-binding activity and the AP-1-mediated transactivation of the reporter construct TRE-CAT between bcl-2-expressing cells and control cells. When compared with control cells, bcl-2-transfected cells expressed significantly more c-fos but not c-jun after TPA treatment. Furthermore, the levels of AP-1 and AP-1-induced transactivation of TRE-CAT were greater in bcl-2-transfected cells than in control cells after TPA treatment. These results showed that bcl-2 cooperates with a tumor promoter such as TPA in the induction of malignant transformation in mouse epidermal cells and that bcl-2 enhances soft-agar growth by stimulating signaling pathways that led to increased AP-1 expression.

Previous studies have demonstrated a role for the fos gene in promoting malignant conversion of mouse skin tumors. In the study reported here, differential display was performed to identify fos- and jun-regulated genes that are differentially expressed during premalignant progression. Total RNA isolated from variants of the papilloma cell line SP-1 transduced with retroviral vectors expressing v-jun and v-fos alone or in tandem was analyzed for the presence of differentially expressed transcripts by using 35 different primer combinations. Differentially expressed clones were rescreened by dot-blot analysis by using cDNA from chemically induced tumors with a high or low risk of malignant conversion. Three differentially displayed fragments were isolated in this analysis. Homology searches indicated that these fragments shared significant homology with the apoptosis inhibitor bcl-2, human alternative splicing factor/splicing factor 2 (ASF/SF2), and a novel gene not present in the GenBank or EMBL databases. In situ hybridization indicated that the expression levels of the bcl-2 homolog increased with malignant potential in chemically derived mouse skin tumors. A similar analysis indicated that expression of the ASF/SF2 homolog was greater in papillomas than in normal skin or in squamous cell carcinomas. Transcripts for this gene were most abundant in the granular layer. The expression pattern of the third differential display fragment was consistent with that of a tumor suppressor gene. This gene was expressed at very high levels in normal skin and benign papillomas but was essentially undetectable in squamous cell carcinomas. Through this approach, we identified known and novel genes that may contribute to malignant progression in epidermal tumors.

Previous studies have demonstrated that overexpression of urinary plasminogen activator (uPA) in rat prostate cancer cells results in increased skeletal metastases, which are primarily of the osteoblastic variety. The osseous activation induced by the metastases appears to be mediated through the amino terminal fragment (ATF) of uPA, which lacks the catalytic domain and can act as a growth factor for osteoblasts. To explore further the mechanism of action of uPA in bone cells, we evaluated the effects of ATF on modulating the expression of various proto-oncogenes. Human-osteoblast-derived osteosarcoma cells, SaOS2, were treated with graded doses of ATF for 10-120 min, and effects on early response proto-oncogenes were monitored. ATF increased c-myc, c-jun, and c-fos gene expression in a time-dependent manner for up to 60 min, after which mRNA levels fell. The maximum induction was seen in c-fos gene expression, which was found to be dose dependent. This effect of ATF was localized to its growth-factorlike domain. Examination of the half life of these transcripts in the presence of the transcriptional inhibitor actinomycin D demonstrated that ATF does not alter the stability of c-fos mRNA in these bone cells. Nuclear run-off assays indicated that ATF effects were due to stimulation of c-fos gene transcription. An increase in c-fos protein levels was correlated with the augmentation of its mRNA in ATF-treated SaOS2 cells. Pretreatment of SaOS2 cells with the protein tyrosine kinase inhibitor herbimycin and recombinant soluble uPA receptor (uPAR) caused a significant reduction in the ability of ATF to induce c-fos expression. These results demonstrate a novel role for uPA in activating early response proto-oncogenes, in particular c-fos, which plays an important role in bone cell growth and differentiation and may be a key factor in the signal transduction pathway of ATF.

Using in situ hybridization histochemistry, we investigated the effects of whisker stimulation in freely moving rats on the expression of the immediate-early genes zif 268 and c-fos in the barrel cortex. Whiskers equipped with metal filaments were stimulated for 5-15 minutes with a pulsating magnetic field. Such whisker stimulation resulted in increased zif 268 and c-fos expression that was largely restricted to radial columns across the barrels representing the stimulated whiskers. In these columns, gene expression was elevated, to a variable degree, across the entire cortical thickness, with a distinct maximum in layer IV. The magnitude of gene expression in a barrel was proportional to the intensity of stimulation. Cellular analysis confirmed that whisker stimulation induced c-fos expression mostly in stellate cells of layer IV and in some pyramidal cells in other layers. However, even after the strongest stimulation, only subsets of neurons were labeled in all layers, suggesting that subpopulations of neurons with a differential genomic response to sensory input exist. These results indicate that the expression of these immediate-early genes is regulated by normal neuronal activity under physiological conditions, and suggest that such gene regulation is an integral part of neuronal function.

There is a close interaction between the processes involved in osteogenesis and hemopoiesis. In developing bone, the osteoclasts, cells of hemopoietic origin, resorb and invade the calcified cartilage rudiment. As a result, the primitive marrow cavity is formed and hemopoiesis initiates. Osteogenic cells-osteoblasts and osteocytes-control the development and activity of the osteoclasts through the local release of factors. One factor responsible for this osteoblast-osteoclast interaction is colony-stimulating factor-1 (CSF-1). Studies performed on the osteopetrotic op/op mouse mutant have established that this factor is essential for proliferation and differentiation of the osteoclasts. Expression of CSF-1 receptors by mature osteoclasts and osteoclast precursors strongly suggests that CSF-1 action is exerted directly on cells of this lineage. In vivo, CSF-1 synthesis by osteoblasts is temporally and spatially related to sites of osteoclast development. Thus CSF-1 may represent one of the factors responsible for coupling hemopoiesis to osteogenesis. In vitro, osteoblasts express at least 4 transcripts encoding either a secreted or a membrane-bound form of CSF-1. At the protein level, osteoblasts in vitro synthesize the membrane-bound form and secrete the majority of CSF-1 as a proteoglycan, a small fraction of which is integrated into the matrix. These different molecular forms may locally restrict the biological action of this cytokine. Indeed, injection of recombinant human CSF-1 in op/ op mutants does not correct the osteoclast deficiency in the metaphyseal spongiosa of long bones, and sclerosis persists at this site. Similarly, the deficiency of some tissue macrophage populations in op/op mice is only partially or not at all corrected by injection of CSF-1. The expression of CSF-1 receptors by mature osteoclasts may imply that CSF-1 also influences their bone resorbing activity. Indeed, CSF-1 has been shown to induce osteoclast fusion, spreading, and survival. These findings suggest that CSF-1 is essential for the proliferation, differentiation, activity, and survival of tissue macrophages and osteoclasts, cells involved in tissue turnover. Furthermore, they corroborate the view that both osteoclasts and tissue macrophages stem from a CSF-1-dependent common precursor along the macrophage lineage.

Potassium ferricyanide is known to elicit cell growth and mitogenesis in various cells by stimulating a transplasma membrane electron-transport system. When serum-starved PC12 cells were treated with potassium ferricyanide, stimulation of mitogenesis was evidenced by enhanced DNA synthesis, as well as by increased cell numbers. Intracellular pH (pH(i)) of PC12 cells was measured at 37 degrees C by microfluorimetric analysis of 2',7'-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). The resting pH(i) of unstimulated cells was 7.52 (external pH 7.40). Addition of potassium ferricyanide (100 microM) decreased pH(i) by about 0.25 pH units. Lowering pH(i) to a similar extent, either by decreasing external pH (pH(o)) or by adding a weak acid, also elicited a mitogenic response, indicating that intracellular acidification by itself has growth factor-mimicking, mitogenic effects. Nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) triggered proliferation without changes in pH(i). The mitogenic treatments eliciting intracellular acidification did not activate protein kinase C (PKC) but stimulated the p42/p44 mitogen-activated protein (MAP) kinase. Our results indicate that 2 distinct mitogenic pathways are active in PC12 cells: the first is independent of pH(i) and involves activation of the PKC pathway and the second requires a permissive pH(i) value around 7.25 and involves activation of the p42/p44 MAP kinase pathway.

Glucocorticoids inhibit the growth and promote the differentiation of normal lung cells. Transformed A5 mouse lung cells containing an activated Ki-ras gene are not responsive to glucocorticoid-induced growth inhibition and demonstrate increased cell proliferation. Activated ras genes may lead to constitutive activation of genes, such as the activating protein 1 (AP-1) transcription factor components fos and jun, which are downstream in the ras signal-transduction pathway. A5 cells and A5GR, a stable A5 transfectant containing excess copies of the glucocorticoid receptor (GR) gene, were examined for potential alterations in AP-1 that accompany the restoration of glucocorticoid-dependent growth inhibition. The established ability of the GR to antagonize AP-1 activity led us to examine the regulation and inducibility of c-fos and c-jun in these cells. Nontransformed C10 lung cells were found to have higher and more inducible AP-1 activity than the transformed A5 cells. The level of AP-1 activity could be reduced in C10 cells by transient transfection of constitutive fos and jun expression vectors. In A5 cells, stimulation with factors that activate the serum-response element on the fos promoter and induce c-fos mRNA had little effect on AP-1 activity, whereas treatment with 12-O-tetradecanoylphorbol-13-acetate, which acts at the fos-AP-1 binding sequence site on the fos promoter, efficiently induced c-fos mRNA. The c-fos mRNA in A5GR cells, however, was not inducible with all treatments, suggesting that one potential mechanism by which the GR restores glucocorticoid-induced growth inhibition in these cells may involve the desensitization of additional 12-O-tetradecanoylphorbol-13-acetate-inducible elements of the fos promoter.

We investigated the structure and the expression of various oncogenes in three of the most common human bone tumors-osteosarcoma (36 samples from 34 patients), giant cell tumor (10 patients), and chondrosarcoma (18 patients)-in an attempt to identify the genetic alterations associated with these malignancies. Alterations of RB and p53 were detected only in osteosarcomas. Alterations of c-myc, N-myc, and c-fos were detected in osteosarcomas and giant cell tumors. Ras alterations (H-ras, Ki-ras, N-ras) were rare. Chondrosarcomas did not contain any detectable genetic alterations. Our results suggest that alterations of c-myc, N-myc, and c-fos oncogenes occur in osteosarcomas, in addition to those previously described for the tumor suppressor genes RB and p53. Moreover, statistical analyses indicate that c-fos alterations occur more frequently in osteosarcoma patients with recurrent or metastatic disease.

The superior cervical ganglion (SCG) is thought to be a good model for correlation studies of morphology, function and metabolism of neurons. The SCG has a relatively simple organization, it can be easily manipulated in situ, and it maintains synaptic transmission and a high metabolic rate during in vitro incubations. The histology and structure of SCG neurons have been characterized in detail, and physiologic stimuli, injury and aging have all been found to induce changes in the SCG morphology. During the last decade, research in the field of signal transduction has greatly expanded. Several signal transduction pathways have been identified that participate in the regulation of neurotransmitter synthesis, gene expression, neuronal excitability and growth factor responses of sympathetic neurons. We have been interested in using the SCG to study some of the second and third messengers involved in converting external stimuli received by sympathetic neurons into cellular short- and long-term events. Using immunohistochemistry, we have investigated protein kinase C-subtypes and the immediate early gene product Fos in the SCG, and characterized some of the changes induced by injury and aging in these messenger molecules. We will review the results and discuss the advantages and disadvantages of using histological methods in the study of signal transduction in sympathetic neurons.

It has long been known that heme oxygenase (HO) is a key enzyme in heme catabolism and recently it was also found to acts as an oxidative stress protein to produce carbon monoxide (CO), which has similar actions to those of nitrogen monoxide (NO). Therefore, we examined transcriptional control of the HO gene in mouse M1 (myeloleukemia) cells during their differentiation into macrophages. Since the promoter region of this gene is known to have a TPA-responsive element (TRE), its expression might be regulated by a C-kinase signal transduction pathway. Then we investigated the activation of the HO gene after treatment of M1 cells with TPA and inhibitors of C-kinase. When M1 cells were treated with TPA, they differentiated into macrophage-like cells. Upon treatment with TPA, H2O2 was produced first, the nuclear proto-oncogenes fos and jun were activated, and then the HO gene was activated. The extent of transcriptional activation of the fos, jun, and HO genes in M1 cells treated with TPA was reduced by a specific inhibitor of C-kinase and a scavenger of oxygen radicals. When M1 cells were treated with H2O2, essentially the same level of transcription of the HO gene was observed, but the extent of transcriptional activation of the fos and jun genes was about half of the treatment with TPA. Super-shift assays using the TRE of the HO gene revealed that the Fos and Jun proteins from nuclei of M1 cells treated with TPA bound to the TRE, and same assays using DNA with the NF-kappa B motif also revealed that the active NF-kappa B protein from M1 cells treated with H2O2 or TPA also bound to the corresponding motif. These results strongly suggest that the HO gene in M1 cells is activated by TPA through a production of H2O2, an oxidative activation pathway of NF-kappa B, and a signal-transduction pathway that involves C-kinase during the differentiation of macrophages that occurs upon treatment with TPA.

The rat tracheal implant model was used to characterize the role of activated Ha-ras in the neoplastic progression of heterogeneous rat tracheal epithelial (RTE) cell populations. An activated Ha-ras-containing cell line, RTE 2-2, and its subclone, RTE 2-2n, which possesses only Ha-ras proto-oncogene alleles, were studied to determine whether activated ras could interact with the downstream signal transduction targets fos and myc and alter their cell-cycle-dependent expression in vitro. Transformed RTE cell lines with activated Ha-ras displayed earlier fos expression, with a peak at 15 min after serum stimulation. These cell lines also displayed a more accelerated loss of fos mRNA than seen in cells without activated Ha-ras. The effects on fos expression kinetics were seen only in cell lines with activated ras and were not related to the transformed phenotype of the cells. No change in myc expression kinetics were observed in any RTE cell line. These results suggest that mutations in ras can lead to alterations in nuclear components of the ras signaling pathway at the level of gene transcription.

In an attempt to find a common DNA alteration occurring in human glioma, we examined DNA methylation in 34 gliomas of various pathological grades and compared them with those in normal cerebral subcortex DNA. The total methylated cytosine levels in the genome did not differ appreciably between the tumors and the normal tissues; however, the degree of DNA methylation in several proto-oncogenes and suppressor oncogenes showed some alterations. Among them, the c-fos gene demonstrated deviation from that of normal tissues in all cases examined, suggesting that the alteration of c-fos gene methylation plays a role in the early steps of human glioma development.

The promoter region of the c-fos oncogene 5' flanking sequence contains enhancer elements crucial for binding nuclear factors that regulate transcription following cell proliferation and differentiation. Single-stranded deoxyoligonucleotides were chosen for modulation of c-fos protooncogene expression because of their high-affinity binding to specific nucleotide sequences. We designed two oligonucleotides that form a triple-helix complex on the retinoblastoma gene product-responsible element of the c-fos oncogene. Modification of the DNA triplex with dimethyl sulfate and affinity cleaving assays demonstrate that the predicted oligonucleotides form a DNA triplex structure with the c-fos promoter in a sequence-specific manner. Tumorigenic and non-tumorigenic fibroblasts were transiently transfected with fos-CAT plasmid modified with alkylating triplex-forming oligonucleotide reagents. A dramatic depression of CAT activity was found when the cross-linked triple helix complex at the retinoblastoma gene product-related site of the c-fos promoter was used. These experiments suggest that transcription of individual genes can be selectively modulated in cell culture by sequence specific triplex formation in regulatory enhancer sequences.

We have studied the expression and activity of the jun and fos families of transcription factors in a panel of human breast cancer cells. Numerous breast-cancer cell lines showed variable levels of expression of jun and fos family-member RNA, activator protein-1 (AP-1) DNA binding, and transcriptional-activating activities during exponential growth. In all of the breast-cancer lines tested, c-jun RNA and AP-1 DNA-binding activity correlated. In addition, in most breast cancer cell lines AP-1 DNA-binding activity also correlates with AP-1-transactivating activity. However, some breast cancer cell lines have high c-jun RNA expression, high AP-1 DNA-binding activity, and low AP-1-transactivating activity. Such results suggest that in these breast cancer cell lines there exist AP-1 complexes that can bind DNA but cannot activate transcription. Multiple peptide growth factors as well as the tumor promoter 12-0-tetradecanoylphorbol-13-acetate induced the expression of jun and fos family-member RNAs and also increased AP-1 DNA-binding activity and functional AP-1-transcriptional activating activity in MCF7 breast cancer cells. However, treatment with estrogen, a steroid growth factor, failed to increase jun and fos RNA expression and induced minimal increases in AP-1 DNA binding and AP-1-induced transcriptional-activating activity in comparison with that seen after peptide hormone treatment. Thus, mitogenic peptide hormones and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate, but not estrogen, strongly activate the AP-1 transcription factor in breast cancer cells. A dominant-negative mutant of c-jun that specifically inhibits AP-1- transactivating activity in rat fibroblasts inhibited AP-1 transactivating activity in breast-cancer cells and blocked the increase in AP-1-mediated transcription induced by serum or specific growth factors. This dominant-negative mutant also inhibited MCF7 colony formation, indicating that expression of this AP-1 inhibitor suppressed the proliferation of these breast cancer cells. Such results suggest that growth factor-induced proliferation of breast cancer cells can possibly be blocked by inhibiting AP-1-transactivating activity.

The ultraviolet light (UV)-responsive element (URE) is an octamer sequence, TGACAACA, that shares homology with cyclic AMP-responsive element and activator protein 1 target sequences. Because URE-binding proteins have been shown to play a role in cellular response to DNA damage, we determined their expression and DNA-binding activities in repair-deficient cells. Of the complementation groups tested, only xeroderma pigmentosum (XP)-C cells induced expression of c-jun after UV irradiation; this correlated with XP-C binding to the URE and resembled the pattern observed with normal human fibroblasts. In other cases either a decrease (XP-A) or no change (XP-D) in URE-binding activities was noticed, which may be associated with decreased c-fos and poor c-jun expression after UV irradiation. That XP-C cells were the only complementation group exhibiting URE-binding activities similar to those of repair-proficient cells points to the possible correlation between proper repair of transcriptionally active genes and the expression and activities of proteins implicated in the cellular response to UV irradiation.

C3H/HeJ (C3H) mice are approximately 50-fold more susceptible to liver-tumor induction than C57BL/6J (B6) mice. This difference is susceptibility is a consequence of allelic differences in hepatocarcinogen sensitivity (Hcs) genes that control the growth of preneoplastic lesions in the liver. We have shown previously that these two strains differ in their responses to partial hepatectomy, which acts as a promoter of hepatocarcinogenesis in B6 mice but not in C3H mice. To determine whether there are also strain-specific differences in normal regulation of hepatic growth, we compared liver regeneration in C3H and B6 mice at the levels of DNA synthesis and gene expression. Partial hepatectomy induced a cascade of controlled events resulting in the regeneration of the liver to its original mass 11 d after surgery. We observed a two-fold greater level of DNA synthesis in C3H mice relative to B6 mice during the first peak of DNA synthesis, which occurred 35 h after hepatectomy in both strains. While the c-fos transcript was readily induced in both strains, there was a reduction in the expression of the late response genes E2F1 and dihydrofolate reductase in the livers of B6 mice when compared with the expression of these transcripts in the livers of C3H mice. The differential regulation of E2F1 between B6 and C3H mice may indicate that the Hcs genes and E2F1 function in the same signal transduction pathway of normal growth control.