Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.

Fibronectin, a plasma protein immunologically identical with a major surface protein of normal fibroblasts, was found to bind to collagen and gelatin. A solid phase enzyme immunoassay was used for the binding tests. Collagen, gelatin or various control proteins were adsorbed to a plastic surface. Binding of fibronectin was detected using purified fibronectin antibodies conjugated to alkaline phosphatase. Circulating fibronectin and fibronectin obtained from fibroblast cultures both showed specific binding to collagen and gelatin. Preparative affinity chromatography of plasma on gelatin coupled to Sepharose gave electrophoretically and immunologically pure fibronectin in high yields. Malignantly transformed fibroblasts lack surface fibronectin. Our findings suggest the possibility that this results in a lack of anchorage to the surrounding intercellular matrix, which could contribute to the malignant growth behavior.

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.

As normal cells progress toward malignancy, they must switch to an angiogenic phenotype to attract the nourishing vasculature that they depend on for their growth. In cultured fibroblasts from Li-Fraumeni patients, this switch was found to coincide with loss of the wild-type allele of the p53 tumor suppressor gene and to be the result of reduced expression of thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis. Transfection assays revealed that p53 can stimulate the endogenous TSP-1 gene and positively regulate TSP-1 promoter sequences. These data indicate that, in fibroblasts, wild-type p53 inhibits angiogenesis through regulation of TSP-1 synthesis.

Various factors are known to regulate cell growth and differentiation, but less is known of agents which affect movement and positioning, particularly in epithelial-mesenchymal interactions. Cultured human embryo fibroblasts release a protein with a relative molecular mass (Mr) of approximately 50,000 (50K) that affects epithelial cells by causing a disruption of junctions, an increase in local motility and a scattering of contiguous sheets of cells. To investigate specificity, a range of cells has been examined for the ability to produce the factor and for sensitivity to its action. Most freshly isolated normal epithelia and epithelia from cell lines of normal tissue, but not epithelia from tumour cell lines or fibroblasts, were sensitive to scatter factor. In contrast, production of the factor, as identified by activity and by chromatography, was restricted to embryonic fibroblasts and certain variants of 3T3 and BHK21 cells and their transformed derivatives. We conclude that the scatter factor is a paracrine effector of epithelial-mesenchymal interaction, which affects the intercellular connections and mobility of normal epithelial cells. The factor might be involved in epithelial migration, such as occurs in embryogenesis or wound healing.

We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.

Antisera against murine Son of sevenless (Sos) recognize a protein of M(r) 155,000 in rat-1 fibroblasts with specific guanine nucleotide exchange activity toward p21c-Ha-ras. Epidermal growth factor (EGF) receptor coimmunoprecipitates with Sos from EGF-stimulated, but not quiescent, cells. The SH2 and SH3 domain-containing "adapter" protein Grb2 is also found in Sos immunoprecipitates in an EGF-inducible manner. In vitro reconstitution shows that Grb2 is required for the binding of activated EGF receptor to Sos. A phosphopeptide corresponding to tyrosine 1068 of the EGF receptor blocks both the assembly of the complex and EGF stimulation of nucleotide exchange on p21ras in a permeabilized cell system. These results suggest that EGF-induced activation of nucleotide exchange on p21ras proceeds through the recruitment of cytosolic Sos to a complex with EGF receptor and Grb2 at the plasma membrane.

A gene has been identified that is expressed specifically in stromal cells surrounding invasive breast carcinomas. On the basis of its sequence, the product of this gene, named stromelysin-3, is a new member of the family of metalloproteinase enzymes which degrade the extracellular matrix. The suggestion is that stromelysin-3 is one of the stroma-derived factors that have long been postulated to play an important part in progression of epithelial malignancies.

We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.

The treatment of cultured rat-1 fibroblasts or H35 hepatoma cells with high concentrations of serum induces the circadian expression of various genes whose transcription also oscillates in living animals. Oscillating genes include rper1 and rper2 (rat homologs of the Drosophila clock gene period), and the genes encoding the transcription factors Rev-Erb alpha, DBP, and TEF. In rat-1 fibroblasts, up to three consecutive daily oscillations with an average period length of 22.5 hr could be recorded. The temporal sequence of the various mRNA accumulation cycles is the same in cultured cells and in vivo. The serum shock of rat-1 fibroblasts also results in a transient stimulation of c-fos and rper expression and thus mimics light-induced immediate-early gene expression in the suprachiasmatic nucleus.