Systemic Metabolic Signatures of Oral Diseases.

Systemic metabolic signatures of oral diseases have been rarely investigated, and prospective studies do not exist. We analyzed whether signs of current or past infectious/inflammatory oral diseases are associated with circulating metabolites. Two study populations were included: the population-based Health-2000 (n = 6,229) and Parogene (n = 452), a cohort of patients with an indication to coronary angiography. Health-2000 participants (n = 4,116) provided follow-up serum samples 11 y after the baseline. Serum concentrations of 157 metabolites were determined with a nuclear magnetic resonance spectroscopy-based method. The associations between oral parameters and metabolite concentrations were analyzed using linear regression models adjusted for age, sex, number of teeth, smoking, presence of diabetes, and education (in Health-2000 only). The number of decayed teeth presented positive associations with low-density lipoprotein diameter and the concentrations of pyruvate and citrate. Negative associations were found between caries and the unsaturation degree of fatty acids (FA) and relative proportions of docosahexaenoic and omega-3 FAs. The number of root canal fillings was positively associated with very low-density lipoprotein parameters, such as diameter, cholesterol, triglycerides, and number of particles. Deepened periodontal pockets were positively associated with concentrations of cholesterol, triglycerides, pyruvate, leucine, valine, phenylalanine, and glycoprotein acetyls and negatively associated with high-density lipoprotein (HDL) diameter, FA unsaturation degree, and relative proportions of omega-6 and polyunsaturated FAs. Bleeding on probing (BOP) was associated with increased concentrations of triglycerides and glycoprotein acetyls, as well as decreased proportions of omega-3 and omega-6 FAs. Caries at baseline predicted alterations in apolipoprotein B-containing lipoproteins and HDL-related metabolites in the follow-up, and both caries and BOP were associated with changes in HDL-related metabolites and omega-3 FAs in the follow-up. Signs of current or past infectious/inflammatory oral diseases, especially periodontitis, were associated with metabolic profiles typical for inflammation. Oral diseases may represent a modifiable risk factor for systemic chronic inflammation and thus cardiometabolic disorders.

Tomato thymidine kinase-based suicide gene therapy for malignant glioma--an alternative for Herpes Simplex virus-1 thymidine kinase.

Malignant gliomas (MGs) are the most common malignant primary brain tumors with a short life estimate accompanied by a marked reduction in the quality of life. Herpes Simplex virus-1 thymidine kinase ganciclovir (HSV-TK/GCV) system is the best characterized enzyme prodrug therapy in use. However, lipophobicity of GCV and low enzymatic activity of HSV-TK reduce the treatment efficacy. Tomato TK (ToTK) has shown high activity in combination with its specific substrate azidothymidine (AZT). The aim of this study was to evaluate whether ToTK/AZT could be used as an alternative to HSV-TK/GCV therapy. Both treatments demonstrated cytotoxicity in human MG cells in vitro. In vivo, both treatments decreased tumor growth and tumors were smaller in comparison with controls in mouse orthotopic MG model. Survival of ToTK/AZT-treated mice was significantly increased compared with control mice (*P<0.05) but not as compared with HSV-TK/GCV-treated mice. No significant differences were observed in clinical chemistry safety analyses. We conclude that both treatments showed a beneficial treatment response in comparison to controls on tumor growth and ToTK/AZT also on survival. There were no significant differences between these treatments. Therefore ToTK/AZT could be considered as an alternative treatment option for MG because of its favorable therapeutic characteristics.

Improved therapeutic effect on malignant glioma with adenoviral suicide gene therapy combined with temozolomide.

Malignant gliomas (MGs) are cancers with poor prognosis and limited therapeutic options. Herpes Simplex virus-1 thymidine kinase expressed from adenoviruses with prodrug ganciclovir (TK/GCV) is the best-characterized suicide gene therapy, whereas temozolomide (TMZ) is the first-line chemotherapy for MG. However, the potential of their combination has not been studied thoroughly. The aim of this study was to evaluate the therapeutic response of this combination and to study whether addition of valproic acid (VPA) could benefit the treatment outcome. Efficacies of different treatments were first studied in vitro in BT4C rat MG cells. Therapeutic assessment in vivo was done in an immunocompetent rat MG model for treatment efficacy and toxicity. In vitro, VPA was able to significantly enhance cytotoxicity and increase adenovirus-mediated transduction efficiency up to sevenfold. In vivo, rats receiving TK/GCV+TMZ had notably smaller tumors and enhanced survival (P<0.001) in comparison with control rats. However, VPA was not able to further enhance the treatment response in vivo. Leukocytopenia and thrombocytopenia were the major side effects. We conclude that careful optimization of the treatment schedules and doses of individual therapies are necessary to achieve an optimal therapeutic effect with TK/GCV+TMZ combination. No further in vivo benefit with VPA was observed.

Epidermal stem cells: location, potential and contribution to cancer.

Epidermal stem cells have been classically characterized as slow-cycling, long-lived cells that reside in discrete niches in the skin. Gene expression studies of niche-resident cells have revealed a number of stem cell markers and regulators, including the Wnt/beta-catenin, Notch, p63, c-Myc and Hedgehog pathways. A new study challenges the traditional developmental paradigm of slow-cycling stem cells and rapid-cycling transit amplifying cells in some epidermal regions, and there is mounting evidence to suggest that multi-lineage epidermal progenitors can be isolated from highly proliferative, non-niche regions. Whether there is a unique microenvironment surrounding these progenitors remains to be determined. Interestingly, cancer stem cells derived from epidermal tumours exist independent of the classic skin stem cell niche, yet also have stem cell properties, including multi-lineage differentiation. This review summarizes recent studies identifying the location and regulators of mouse and human epidermal stem cells and highlights the strategies used to identify cancer stem cells, including expression of normal epidermal stem cell markers, expression of cancer stem cell markers identified in other epidermal tumours and characterization of side-population tumour cells.

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy.

Cancer suicide gene therapy based on herpes simplex virus type I thymidine kinase (HSV-TK) and ganciclovir (GCV) suffers from the lack of efficacy in clinical use, which is mostly due to low gene-transfer efficiency and absence of bystander effect in tumors. We have previously demonstrated the enhancement of GCV cytotoxicity by fusing the HSV-TK with the cell penetrating peptide from HIV-1 transactivator protein transduction domain (TAT PTD). Despite the earlier promising results, we found that the triple fusion protein HIV-1 transactivator protein transduction domain-thymidine kinase suicide gene-green fluorescent protein marker gene (TAT-TK-GFP) increased GCV cytotoxicity only in 3/12 of different human tumor cell lines. Extended GCV exposure enhanced the cytotoxic effect of HSV-TK/GCV gene therapy, but the difference between TK-GFP and TAT-TK-GFP was not statistically significant. The modest improvement on cell killing mediated by TAT PTD in Chinese hamster ovary cells appeared to be associated with cell-surface heparan sulfate proteoglycan (HSPG) composition. However, TAT-mediated increased cell death did not correlate with the density of cell-surface HSPG expression in different tumor cell lines. In conclusion, although some degree of enhancement by TAT was shown in certain tumor cells in vitro, it is unlikely that TAT peptide linked to a suicide protein could be a useful booster of in vivo gene therapy trials.

Gene targeting of envoplakin, a cytoskeletal linker protein and precursor of the epidermal cornified envelope.

Envoplakin, a member of the plakin family of cytoskeletal linker proteins, is localized in desmosomes of stratified epithelial cells and is a component of the epidermal cornified envelope. Gene targeting in mouse embryonic stem cells was used to generate a null allele of envoplakin. No envoplakin transcripts from the targeted allele could be detected in the skin of newborn mice. Mice homozygous for the targeted allele were born in the normal Mendelian ratio and were fertile. They did not develop any discernible pathological phenotype up to the age of 1 year. The ultrastructural appearance of cornified envelopes from adult epidermis was indistinguishable between wild-type and knockout mice, and there was no evidence that the absence of envoplakin affected the subcellular distribution of periplakin or desmoplakin, two other plakins found in desmosomes. The proportion of immature cornified envelopes in the epidermis of newborn mice was greater in envoplakin-null animals than in heterozygous littermates or wild-type mice, and the envelopes had a larger surface area. This correlated with a slight delay in barrier acquisition during embryonic development. We conclude that although envoplakin is part of the scaffolding on which the cornified envelope is assembled, it is not essential for envelope formation or epidermal barrier function.

Subcellular distribution of envoplakin and periplakin: insights into their role as precursors of the epidermal cornified envelope.

Envoplakin and periplakin are two plakins that are precursors of the epidermal cornified envelope. We studied their distribution and interactions by transfection of primary human keratinocytes and other cells. Full-length periplakin localized to desmosomes, the interdesmosomal plasma membrane and intermediate filaments. Full length envoplakin also localized to desmosomes, but mainly accumulated in nuclear and cytoplasmic aggregates with associated intermediate filaments. The envoplakin rod domain was required for aggregation and the periplakin rod domain was necessary and sufficient to redistribute envoplakin to desmosomes and the cytoskeleton, confirming earlier predictions that the proteins can heterodimerize. The linker domain of each protein was required for intermediate filament association. Like the NH(2) terminus of desmoplakin, that of periplakin localized to desmosomes; however, in addition, the periplakin NH(2) terminus accumulated at cell surface microvilli in association with cortical actin. Endogenous periplakin was redistributed from microvilli when keratinocytes were treated with the actin disrupting drug Latrunculin B. We propose that whereas envoplakin and periplakin can localize independently to desmosomes, the distribution of envoplakin at the interdesmosomal plasma membrane depends on heterodimerization with periplakin and that the NH(2) terminus of periplakin therefore plays a key role in forming the scaffold on which the cornified envelope is assembled.

Structure and regulation of the envoplakin gene.

Envoplakin, a member of the plakin family of proteins, is a component of desmosomes and the epidermal cornified envelope. To understand how envoplakin expression is regulated, we have analyzed the structure of the mouse envoplakin gene and characterized the promoters of both the human and mouse genes. The mouse gene consists of 22 exons and maps to chromosome 11E1, syntenic to the location of the human gene on 17q25. The exon-intron structure of the mouse envoplakin gene is common to all members of the plakin family: the N-terminal protein domain is encoded by 21 small exons, and the central rod domain and the C-terminal globular domain are coded by a single large exon. The C terminus shows the highest sequence conservation between mouse and human envoplakins and between envoplakin and the other family members. The N terminus is also conserved, with sequence homology extending to Drosophila Kakapo. A region between nucleotides -101 and 288 was necessary for promoter activity in transiently transfected primary keratinocytes. This region is highly conserved between the human and mouse genes and contains at least two different positively acting elements identified by site-directed mutagenesis and electrophoretic mobility shift assays. Mutation of a GC box binding Sp1 and Sp3 proteins or a combined E box and Krüppel-like element interacting with unidentified nuclear proteins virtually abolished promoter activity. 600 base pairs of the mouse upstream sequence was sufficient to drive expression of a beta-galactosidase reporter gene in the suprabasal layers of epidermis, esophagus, and forestomach of transgenic mice. Thus, we have identified a regulatory region in the envoplakin gene that can account for the expression pattern of the endogenous protein in stratified squamous epithelia.

Extracellular matrix-dependent activation of syndecan-1 expression in keratinocyte growth factor-treated keratinocytes.

Syndecan-1 is a major heparan sulfate proteoglycan of the epidermis. Its expression is strongly induced in migrating and proliferating keratinocytes during wound healing and, on the other hand, diminished or lost in invasive squamous cell carcinoma. We have recently found in the syndecan-1 gene an enhancer (fibroblast growth factor-inducible response element (FiRE)) that activates gene expression in wound edge keratinocytes (Jaakkola, P., Kontusaari, S., Kauppi, T., Määttä, A., and Jalkanen, M. (1998) FASEB J. 12, 959-969). Now, we demonstrate that the activation of this enhancer by keratinocyte growth factor (KGF) is modulated by the components of the extracellular matrix (ECM). MCA-3D mouse immortal keratinocytes growing on fibrillar collagen failed to activate FiRE and subsequently to induce syndecan-1 in response to KGF. The same cells growing on fibronectin or laminin, however, increased FiRE-dependent reporter gene expression upon KGF treatment. The inhibition of the KGF induction by collagen appears to be specific for signaling to FiRE, as the increase in cell proliferation by KGF was not affected. The effect was selective to KGF, as EGF-induction was independent on ECM composition. Changes in the transcription factor binding were not involved in the differential activation of FiRE, as the levels and composition of the AP-1 complexes were unchanged. However, application of anisomycin, an activator of Jun amino-terminal kinase, resulted in a lower response in cells growing on collagen compared with fibronectin. These results indicate that the composition of ECM and availability of growth factors can play a role in the epidermal regulation of syndecan-1 expression and that FiRE is a novel target for gene regulation by the extracellular matrix.

The activation and composition of FiRE (an FGF-inducible response element) differ in a cell type- and growth factor-specific manner.

The expression of the heparan sulfate proteoglycan, syndecan-1, is induced both in keratinocytes and in fibroblasts during development and tissue regeneration. Here we report that in keratinocytes the syndecan-1 gene was stimulated by EGF but not by FGF-2. In fibroblasts it was stimulated by FGF-2 but not by EGF. Likewise, the recently discovered FGF-inducible response element (FiRE) on the gene of syndecan-1 was stimulated by FGF-2 in fibroblasts and by EGF in keratinocytes, but not vice versa. The FiRE has two binding sites for an activator protein-1 (AP-1), one for an FGF-inducible nuclear factor (FIN-1) and one for an upstream stimulatory factor-1 (USF-1). The growth factor-stimulated binding of these transcription factors, as well as their requirement for FiRE activation, varied between the two cell types. First, although AP-1s were required for activation of FiRE in both cell types, the binding of AP-1 to FiRE was increased by growth factor-stimulation only in fibroblasts and not in keratinocytes. Secondly, FiRE did not bind FIN-1 nor needed the FIN-1 binding site for EGF-stimulated activation in keratinocytes, in contrast to the FGF-stimulated activation of FiRE in fibroblasts. Thirdly, EGF, which did not activate FiRE in fibroblasts, failed to activate FIN-1 in these cells. Finally, an USF-1 binding site that was necessary for activation of FiRE in keratinocytes was not needed in fibroblasts. These data suggest mechanisms by which members of the EGF- and FGF-families can differentially stimulate transcription through AP-1 regulated elements in a cell type-specific manner.

Activation of an enhancer on the syndecan-1 gene is restricted to fibroblast growth factor family members in mesenchymal cells.

Fibroblast growth factors (FGFs) induce a variety of biological effects on different cell types. They activate a number of genes, including immediate-early genes, such as the transcription factors Fos and Jun, which are also common targets for other tyrosine kinase receptor-activating growth factors. Here we describe a secondary far-upstream enhancer on the syndecan-1 gene that is activated only by members of the FGF family in NIH 3T3 cells, not by other receptor tyrosine kinase-activating growth factors (e.g., epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, or serum). This FGF-inducible response element (FiRE) consists of a 170-bp array of five DNA motifs which bind two FGF-inducible Fos-Jun heterodimers, one inducible AP-2-related protein, a constitutively expressed upstream stimulatory factor, and one constitutive 46-kDa transcription factor. Mutational analysis showed that both AP-1 binding motifs are required, but not sufficient, for FiRE activation. Moreover, agents such as 12-O-tetradecanoylphorbol-13-acetate, okadaic acid, or forskolin, which are known to activate AP-1 complexes and AP-1-driven promoters, fail to activate FiRE. However, FiRE can be activated by the tyrosine kinase phosphatase inhibitor orthovanadate. Taken together, this data implies a differential activation of growth factor-initiated signaling on AP-1-driven regulatory elements.

Functional characterization of mouse syndecan-1 promoter.

The members of the syndecan family are temporally and spatially expressed heparan sulfate proteoglycans of various tissues, where they mediate extracellular influences on cell morphology and behavior. Functional characterization of the mouse syndecan-1 promoter was carried out in order to elucidate the mechanisms involved in the maintenance of the high transcription levels of syndecan-1 gene in various epithelia. For that 9.5 kilobase pairs of the upstream region of mouse syndecan-1 gene were cloned, sequenced, and used to prepare chimaeric constructs with a reporter gene followed by transient or stable transfections into NMuMG cells, cultured either in the presence or absence of serum, the 2.5-kilobase pair promoter region resulted in the constitutive transcription activity, whereas in 3T3 cells the serum depletion decreased the promoter activity significantly. Deletion of the upstream sequences to -437 base pairs relative to the translation initiation site had little effect on this promoter activity. Further deletion to -365 base pairs removed three GT boxes and slightly increased the promoter activity, whereas the deletion of the next two GC boxes (to -326 base pair) reduced the promoter activity dramatically. All of the GC or GT box sequences bound the same set of Sp1-like nuclear protein in gel shift assays. Nuclear protein binding was also demonstrated around both of the most intense transcription initiation sites. Mutation of these regions separately resulted in total loss of transcription initiation from the deleted site and decreased the promoter activity in relation to the intensity of the abolished start site. This indicates that the transcription initiation of the syndecan-1 gene is directed through initiator-like elements directly overlapping the start sites, as shown for several TATA-less housekeeping and growth regulated genes. We assume that the constitutive high level gene expression in epithelial cells is achieved by the proximal promoter, which is controlled by members of Sp1 transcription factor family.

Effect of the 3'-untranslated region on the expression levels and mRNA stability of alpha 1(I) collagen gene.

Changes in the synthesis of type I collagen, a major extracellular matrix component in skin and bones, are associated with both normal growth or repair processes and with several pathological conditions such as lung fibrosis and liver cirrhosis. The expression of the alpha 1(I) collagen gene is regulated by transcriptional and post-transcriptional mechanisms. Regulation at both these levels are usually utilised when extensive changes occur in collagen synthesis. We constructed plasmids carrying the whole or partially deleted 3'-UTR sequences of the alpha 1(I) collagen gene, fused to two hGH exons and to the promoter of the alpha 1(I) collagen gene. A control plasmid contained the 3'-UTR of the hGH gene. In transient transfections into Rat-1 fibroblasts, no significant differences between plasmids were found, which suggests that although 3'-end of the gene has been shown in previous studies to contain DNaseI hypersensitive sites and to bind sequence-specific nuclear proteins it does not seem to function as a transcriptional regulator. This was further supported by the finding that TGF-beta treatment induced a 2.5-fold expression of hGH mRNA from plasmids containing collagen promoter and either hGH or alpha 1(I) collagen 3'-UTR. In stable transfections, mRNAs using the first polyadenylation site were not as stable as those transcribed from the endogenous alpha 1(I) collagen gene. We suggest that the 3'-UTR alone may not be sufficient to determine the stability of the shorter alpha 1(I) collagen mRNA species.

Extended expression of cartilage components in experimental pseudoarthrosis.

The healing of femoral fractures in an experimental rat pseudoarthrosis model was followed by studying the expression of cartilage specific genes coding for type II and X collagens and aggrecan, soft tissue and bone specific type I collagen, and decorin. Severe impairment of healing was observed with cartilage gene expression continuing until the seventh week and then declining rapidly. The abnormal healing pattern results in an inactive scar-like callus after the ninth week of healing even though house-keeping (e.g., GAPDH) genes are continuously expressed in the tissue. These results could be explained on the basis of continuous chondrogenic stimulus extending much beyond the normal range. If union is not achieved because of mechanical instability, signal of endochondral ossification persists until it becomes exhausted and callus at the fracture gap becomes an inactive fibrous scar. The disturbed matrix gene expression was confirmed by histology.

Nuclear and cytoplasmic alpha 1 (I) collagen mRNA-binding proteins.

We have recently identified a cytoplasmic protein, alpha 1-RBF67, that specifically interacts with the conserved 3'-untranslated region of the alpha 1 (I) collagen gene. The binding activity was decreased in extracts from dexamethasone treated cells, which correlates with the known accelerated turnover of the COL1A1 RNA [Määttä, A. and Penttinen, R.P.K. (1993) Biochem. J. 295, 691-698]. Now we report that a very similar protein is present in nuclear extracts of NIH 3T3, human fibroblast and HeLa cells, which suggests that determination of cytoplasmic mRNA stability is not the sole function of the alpha 1-RBF67 activity. The binding to the RNA probe can be inhibited by annealing a DNA oligonucleotide or using excess of cold specific competitors. In UV-cross linking assays the nuclear protein has the same molecular weight (67 kDa) as the cytoplasmic one and the RNA-bound peptides generated by CNBr or V8 protease cleavage from both the cytoplasmic and the nuclear protein were identical. This protein was the only one of several nuclear collagen mRNA 3'-UTR binding proteins that was present in both nuclear and cytoplasmic extracts. In fibroblasts heparin-resistant nuclear RNA binding proteins had molecular weights of 45, 67 (alpha 1-RBF67), and 71 kDa. HeLa-cells contained an additional protein of 51 kDa and several non-specific RNA-binding proteins. The binding activity is modified by changes in the redox state, which implicates that in the nucleus the binding affinities of alpha 1(I) collagen RNA-binding protein and AP-1, a redox sensitive nuclear factor, that is important in the transcription of alpha 1(I) collagen gene, can be regulated simultaneously to the same direction.

Fibroblast expression of collagens and proteoglycans is altered in aspartylglucosaminuria, a lysosomal storage disease.

Aspartylglucosaminuria (AGU), a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (E.C. 3.5.1.26), is characterised by progressive mental retardation and variable connective tissue signs. The ultrastructure of collagen fibrils in skin of AGU patients is abnormal and their fibroblasts synthesise reduced amounts of collagens [Näntö-Salonen et al. (1984) Lab invest. 51: 464-468]. In this work we measured the steady-state messenger RNA levels of several extracellular matrix components in skin fibroblast cultures of two patients homozygous for the most prevalent mutation (AGUFin) causing the disease in Finland. In confluent cultures the steady-state mRNA concentrations of type I and III collagens were reduced to 0.5-20% of control values. Almost as marked reduction was observed in the mRNA level of biglycan, a small interstitial proteoglycan whereas that of decorin, a closely related, collagen fibril-associated proteoglycan, was increased several-fold. Elevated decorin and decreased biglycan mRNA levels reflected the amounts of the produced corresponding proteoglycans. The differences in the mRNA levels become more pronounced with the time the cells were in culture. Fibronectin mRNA concentrations were similar in AGU and control fibroblasts. Changes in the expression and synthesis of extracellular matrix components might be related to the connective tissue symptoms of the patients.

Suppressed collagen gene expression and induction of alpha 2 beta 1 integrin-type collagen receptor in tumorigenic derivatives of human osteogenic sarcoma (HOS) cell line.

Cell-matrix interactions and intergrin-type cell adhesion receptors are involved in the regulation of tumor cell invasion and metastasis. We have analyzed the expression of matrix proteins and their cellular receptors in human osteosarcoma cells (HOS) and in their virally (KHOS-NP) and chemically (HOS-MNNG) transformed tumorigenic subclones. Transformation decreased dramatically the cellular mRNA levels of alpha 1(I) collagen. Concomitantly with down-regulation of collagen mRNA levels the synthesis of the collagen receptor, alpha 2 beta 1 integrin, was induced. No alpha 2 integrin mRNA was found in HOS cells, suggesting that its expression was regulated most probably at the transcriptional level. 5-Azacytidine alone or combined with alpha 2 integrin-stimulating cytokines, transforming growth factor-beta 1, and interleukin-1 beta, did not turn on the alpha 2 integrin gene. In chemically transformed cells, however, alpha 2 integrin expression could be regulated by cytokines. Thus, we suggest that HOS cells have a strong element, probably other than cell culture-generated de novo promoter methylation, suppressing alpha 2 integrin expression and that this factor is lost in both chemical and viral transformation. Furthermore, the mechanism used by cytokines and malignant transformation to increase alpha 2 integrin expression seems not to be identical. Other transformation-related changes in beta 1 integrins were (i) reduction of the intracellular pool of precursor beta 1 (in HOS-MNNG cells), leading to faster maturation rate of beta 1 subunit and slower maturation rate of alpha subunits, and (ii) decreased electrophoretic mobility of both alpha and beta 1 subunits. At the cellular level both chemical and viral transformation increased cell adhesion to type I collagen.

A fibroblast protein binds the 3'-untranslated region of pro-alpha 1(I) collagen mRNA.

Post-transcriptional regulation of the expression of the pro alpha 1(I) chain of type I collagen (COL1A1) was studied by analysing cytoplasmic RNA-binding proteins and by transient transfections with collagen minigene plasmids. In this paper we present evidence for a factor from NIH 3T3 cells and human skin fibroblasts that interacts with the conserved 3'-untranslated region (UTR) of the shorter 4.8 kb mRNA species of the COL1A1 gene. The specificity of the interaction was confirmed by using (i) unlabelled specific and non-specific competitor RNAs and (ii) oligodeoxyribonucleotides annealed to the probe or used as single-stranded competitors. An antisense oligonucleotide annealed to the RNA probe near its 3'-terminus [20-42 nucleotides upstream of the first polyadenylation signal of the alpha 1(I) collagen mRNA] inhibited the binding, whereas other sense or antisense oligonucleotides had no effect on the interaction. The binding was sensitive to alkylation of free SH groups but not to phosphatase treatment of the extracts. In u.v. cross-linking analysis this factor migrated as a single polypeptide chain of about 67 kDa, and was named alpha 1-RBF67 (type I collagen alpha 1 chain RNA-binding factor). Dexamethasone treatment of fibroblasts, which is known to accelerate the turnover of COL1A1 mRNA, decreased the alpha 1-RBF67 activity markedly as evaluated by gel-retardation and u.v. cross-linking assays. Transient transfections with plasmids carrying the alpha 1(I) collagen promoter and 3'-UTR sequences demonstrated that the 3'-UTR participates in the response to dexamethasone. Thus the loss of alpha 1-RBF67 activity might be associated with decreased alpha 1(I) collagen mRNA levels after dexamethasone treatment.

Nuclear factor binding to an AP-1 site is associated with the activation of pro-alpha 1(I)-collagen gene in dedifferentiating chondrocytes.

Isolated chondrocytes grown on plastic gradually lose their differentiated phenotype upon subculturing. This dedifferentiation is manifested by an altered production of extracellular-matrix molecules (ECM): e.g., the cartilage specific type II collagen is replaced by types I and III. We have studied the regulation of ECM gene expression in dedifferentiating human and murine fetal chondrocytes. Nuclear extracts from dedifferentiated cells, human fetal fibroblasts and 3T3 cells contained a protein that bound in an electrophoretic mobility shift assay to an AP-1 site in the first intron of the human alpha 1(I) collagen gene. This binding activity was not present in freshly isolated human or murine chondrocytes, which produced type II, but not type I, collagen mRNA in culture. Thus the binding activity was induced simultaneously with alpha 1(I)-collagen-gene expression during dedifferentiation. The specific interaction was sensitive to dephosphorylation of the nuclear extract and to chemical modification of reduced cysteine residues. The AP-1 site we studied had previously been shown to be a positive transcriptional contributor in the first intron to the expression of the alpha 1(I) collagen gene. In transient transfections into dedifferentiating chondrocytes, an alpha 1(I) collagen expression plasmid carrying a mutated AP-1 site in the first intron resulted in three-times-lower reporter gene RNA levels than a plasmid carrying the respective functional AP-1 site. These data suggest that the AP-1 sequence and its respective trans-acting factors may play a role in the transcriptional regulation of the alpha 1(I) collagen gene during dedifferentiation of chondrocytes.