Extracellular matrix regulates expression of the TGF-beta 1 gene.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation and modulates the interactions of cells with their extracellular matrix (ECM), in part by inducing the synthesis of various ECM proteins. Three different isoforms of TGF-beta are synthesized in a defined pattern in specific cell populations in vivo. In the specific case of TGF-beta 1, this well-defined and limited expression stands in sharp contrast to its synthesis by virtually all cells in culture. Using mammary epithelial cells as a model system, we evaluated the substratum dependence of the expression of TGF-beta 1. The level of TGF-beta 1 expression is high in cells on plastic, but is strongly downregulated when cells are cultured on a reconstituted basement membrane matrix. In contrast, TGF-beta 2 mRNA levels in cells on either substratum remain unchanged. Using the chloramphenicol acetyl transferase gene as reporter gene under the control of the TGF-beta 1 promoter, we show that transcription from this promoter is suppressed when the cells are in contact with either endogenously synthesized or exogenously administered basement membrane. TGF-beta 1 promoter activity is strongly induced by the absence of basement membrane, i.e., by direct contact of the cells with plastic. This modulation of transcription from the TGF-beta 1 promoter occurs in the absence of lactogenic hormones which allow full differentiation. Our results thus indicate that basement membrane is an important regulator of TGF-beta 1 synthesis, and explain why most cells in culture on plastic express TGF-beta 1 in contrast with the more restricted TGF-beta 1 synthesis in vivo. We propose that there is a feedback loop whereby TGF-beta 1-induced synthesis of basement membrane components is repressed once a functional basement membrane is present. Finally, these results together with our current knowledge of regulation of TGF-beta 1 and TGF-beta 2 synthesis, suggest that, in vivo, TGF-beta 1 may play a major role in regulating the ECM synthesis and the cell-ECM interactions, whereas TGF-beta 2 may be more important in morphogenetic processes.

Laminin mediates tissue-specific gene expression in mammary epithelia.

Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3' untranslated region.

The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.

Characterization of BCE-1, a transcriptional enhancer regulated by prolactin and extracellular matrix and modulated by the state of histone acetylation.

We have previously described a 160-bp enhancer (BCE-1) in the bovine beta-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-beta and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-beta but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.

A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression.

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate beta-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of beta-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes beta-casein unidirectionally into a lumen. We have further shown that ECM- and prolactin-dependent regulations of beta-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine beta-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the beta-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive beta-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECM-mediated regulation.

Regulation of gene expression and cell function by extracellular matrix.

Cells within tissues of multicellular organisms are intimately surrounded by a complex arrangement of extracellular proteins that together act as an informational entity in the sense that they receive, impart, and integrate structural and functional signals. This macromolecular network, composed largely of glycoproteins and proteoglycans, is collectively known as the extracellular matrix (ECM). In this review we aim to show that the ECM plays a fundamental role in both the control of gene expression and the induction and maintenance of tissue-specific function. Also, we revisit a general model that describes how the ECM together with the cytoskeleton and nuclear matrix may regulate tissue-specific gene expression.

Pudendal nerve palsy complicating intramedullary nailing of the femur.

A prospective study of 106 patients who had static interlocking nailing of the shaft of the femur was performed to determine the relationship between the duration and magnitude of intraoperative traction and the development of a pudendal nerve palsy. A strain-gauge, mounted in the countertraction post, measured the magnitude of the perineal pressure over time. All nailings were performed with the patient in the supine position. Postoperatively, the patients were interviewed by one of us, who had been blinded from the results of the recordings of intraoperative pressure, for a history of erectile dysfunction and changes in labial, scrotal, or penile sensation. A light-touch sensory examination of the genitalia was performed on all patients. Ten patients (six men and four women) had a pudendal nerve palsy: nine had sensory changes only, and one complained of erectile dysfunction. The symptoms had resolved at the three-month follow-up evaluation in all patients except one man who complained of dysesthesia six months postoperatively. The patients in whom a palsy did not develop had been positioned on the fracture-table and the perineal post for an average of 2.6 hours (range, 1.4 to 5.2 hours) compared with an average of 2.8 hours (range, 2.0 to 4.3 hours) for those in whom a palsy did not develop (p = 0.15). The magnitude of the total traction forces averaged 34.9 kilogram-hours for the patients who did not have a palsy compared with 73.3 kilogram-hours for those who did (p < 0.03). Adduction of the hip, as well as manipulations for reduction of the fracture, significantly increased the traction forces.(ABSTRACT TRUNCATED AT 250 WORDS)

Lower limb response and injury in frontal crashes.

This article examines two observational and two experimental data sets that emphasize lower limb injuries in passenger car crashes. Statistics show that 60% of moderate-to-severe below-knee injuries sustained by front seat occupants in head-on crashes occur with > 3 cm of footwell intrusion. Moreover, crash tests and computer simulations of car-to-car frontal offset collisions show no causal relationship between the magnitude of footwell intrusion and the axial load measured in the dummy leg. This article correlates below-knee injuries with several factors that influence their frequency and severity, such as the vehicle change in velocity, the magnitude of footwell intrusion, the rate and timing of the intrusion and the size of the vehicle. The vehicle change in velocity and the intrusion rate and timing had the greatest influence on the risk of lower limb injury, while the other factors had much less of an effect.

Combined herbal preparation for topical treatment of Herpes labialis.

BACKGROUND: The efficacy of many preparations for topical use in herpes infections have remained rather disappointing. The development of new antiviral drugs, especially herbal preparations, thus remains desirable. In a screening study with plant extracts, a rhubarb root extract and a sage extract showed a promising activity. OBJECTIVE: The efficacy of a combined topical preparation with rhubarb and sage extracts, of a single-agent preparation with sage extract and of a reference treatment was investigated in a double-blind, comparative, randomised trial. PATIENTS AND METHODS: A total of 149 patients participated, and 145 patients (111 female, 34 male) of whom 64 received the rhubarb-sage cream, 40 the sage cream and 41 Zovirax cream could be evaluated by intention-to-treat analysis. The dried rhubarb extract (23 mg/g) is a standardised aqueous- ethanolic extract according to the German Pharmacopoeia (DAB) with 4.0-6.0% hydroxyanthracene derivatives. The dried sage extract (23 mg/g) is an aqueous extract. The reference product was Zovirax cream (Zovirax(R) Creme) with the active ingredient aciclovir (50 mg/g). RESULTS: The mean time to healing in all cured patients was 7.6 days with the sage cream, 6.7 days with the rhubarb-sage cream and 6.5 days with Zovirax cream. There were statistically significant differences in the course of the symptoms. For the parameter 'swelling', at the 1st followup visit there was a significant advantage for Zovirax cream compared to sage cream, and for the parameter 'pain', at the 2nd follow-up visit there was a significant difference in favour of the rhubarb-sage cream compared to the sage cream. CONCLUSION: The combined topical sage-rhubarb preparation proved to be as effective as topical aciclovir cream and tended to be more active than the sage cream.

Transcriptional activation by viral enhancers: critical dependence on extracellular matrix-cell interactions in mammary epithelial cells.

Extracellular matrix (ECM)-cell interactions are essential for the regulation of many genes in differentiated cell types. A number of expression vectors that work well in cells cultured on tissue-culture plastic appear to be inactive or sporadically active in vivo. We reasoned that these responses also may be influenced by the ECM. We therefore examined three commonly used viral enhancers and found that they all responded either positively or negatively to the presence of exogenous ECM. Using mouse mammary epithelial cells, we found that a mouse mammary tumor virus enhancer linked to its own promoter or to a truncated (and by itself inactive) beta-casein promoter drove transcription efficiently only when the cells were in contact with an ECM (more than a 100-fold induction over tissue-culture plastic). Similarly, the cytomegalovirus enhancer was more active in cells in contact with ECM. In contrast, the simian virus 40 enhancer, linked to the beta-casein promoter, was 12-fold more active in cells on tissue-culture plastic. This activity was strongly reduced when the cells interacted with ECM. Thus, we conclude that different enhancers can respond to ECM by either activating or suppressing transcription. This observation has important implications for understanding the mechanisms of promoter action and for designing expression systems for use in gene therapy.

A mycoplasmal protein influences tumour cell invasiveness and contact inhibition in vitro.

Fab fragments of a monoclonal antibody directed against p37, a protein associated with the surface of FS9 mouse sarcoma cells, were previously found to inhibit the highly invasive behaviour of FS9 cells in vitro. We show that p37 originates from Mycoplasma hyorhinis. Infecting various cell lines with the mycoplasma consistently increased their invasiveness when confronted with chicken heart fibroblasts using Abercrombie's confronted explant technique. Conversely, removing the mycoplasmas or blocking p37 with specific Fab fragments reduced invasiveness. Analysis of individual cell collisions using time-lapse filming showed that the addition of Fab fragments to cells infected with M. hyorhinis greatly increased the level of contact inhibition. The antibody also reduced the invasiveness of transformed cells that did not express the p37 antigen. Hence, a cellular protein or proteins that are structurally related to p37 apparently influence invasive behavior.

Lower extremity injuries in drivers of airbag-equipped automobiles: clinical and crash reconstruction correlations.

OBJECTIVE AND DESIGN: To determine the relationship between airbags and lower extremity injuries, 10 drivers admitted to a level-I trauma center with substantial lower extremity trauma incurred in crashes involving airbag-equipped vehicles were studied in depth with regard to their injuries, the circumstances of the crashes, and the medical charges for the acute management of those injuries. MATERIALS AND METHODS: During the clinical investigation portion of this study, we photographed lower extremity injuries, both soft tissue and radiographs, and performed a detailed surgical exploration during the debridement of open wounds or fracture fixation to treat them appropriately and to define the mechanism of injury, the fracture pattern, the pattern of soft-tissue insult, and the extent of periosteal stripping. We recorded the hospital and professional charges associated with the acute management not only of these injuries, but of the other injuries as well. The analysis performed for each case included a detailed crash reconstruction, including force, contact point, and vehicle intrusion data. Particular attention was paid to the dashboard and toe pan areas to determine deformation and intrusion and their association with thigh, leg, and foot injuries. Pertinent deformation and trajectory information was entered into the Calspan Reconstruction of Accident Speeds on the Highway (CRASH) computer program to generate a delta V or change in velocity measurement used as a measure of collision severity. When field data were incompatible with the limitations of the CRASH program, manual calculations such as "slide to stop" and conservation of momentum formulas were used. RESULTS: The seven male and three female drivers had a mean age of 39.4 years. Only four used seatbelt restraints. The mean delta V was 28.3 mph and the mean maximum crush was 32.4 inches. The mean Injury Severity Score of 13.2. Musculoskeletal injuries included 11 foot/ankle fractures, 6 tibial fractures, 2 patellar fractures, 6 femoral fractures, and two acetabular/pelvic fractures. Other trauma included abdominal, thoracic, head and upper torso injuries, it seems that these safety devices do not prevent injuries to the lower extremity.

Mechanisms of tumour cell metastasis.

Abercrombie and his colleagues have accumulated evidence that changes in the heterotypic contact-inhibition response are largely responsible for the invasiveness of cells, at least in culture. We have identified a 37,000 Mr protein on the surface of mouse fibrosarcoma cells that is involved in their in vitro invasion. Blocking this protein with specific antibodies inhibits the invasion of chicken heart fibroblasts by the tumour cells and normal heterotypic contact inhibition is restored. These results are presented in the general framework of metastatic mechanisms and we review a selection of more recent studies aimed at describing the metastatic phenotype in molecular terms.

A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells.

FS9 mouse sarcoma cells were previously shown to be highly invasive when confronted with chicken heart fibroblasts using Abercrombie's confronted explant technique. This invasion could be inhibited by addition to the assay of Fab fragments of a monoclonal antibody directed against p37, a protein associated with the surface of FS9 cells. We have cloned and sequenced the gene for p37. We show that it originates from Mycoplasma hyorhinis and that UGA is a tryptophan codon in this organism. We present evidence that the p37 gene is part of an operon encoding two additional proteins which are highly similar to components of the periplasmic binding-protein-dependent transport systems of Gram-negative bacteria, and we suggest that p37 is part of a homologous, high-affinity transport system in M. hyorhinis, a Gram-positive bacterium. We discuss the influence of p37 and M. hyorhinis on contact inhibition of locomotion of mammalian cells.

Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

Selective versus non-selective antiarrhythmic approach for prevention of atrial fibrillation after coronary surgery: is there a need for pre-operative risk stratification? A prospective placebo-controlled study using low-dose sotalol.

AIM: This study evaluated the advantages of 'selective' over 'non-selective' antiarrhythmic prevention of atrial fibrillation after coronary surgery based on a new risk prediction algorithm. METHODS AND RESULTS: In a retrospective analysis of a prospective randomized trial, a model for risk prediction was determined based on clinical data of the control group (A; n = 107) and tested in a test group (B; n = 107, treated with low dose sotalol). Using this algorithm, the effect of a 'selective' antiarrhythmic approach in high-risk patients was compared to a 'non-selective' approach, where all patients were treated. In total, 75 (35%) patients developed atrial fibrillation and 14 (7%) side-effects led to discontinuation of study medication. Based on the risk prediction algorithm, 36% of group A patients were classified as high-risk patients with an incidence of atrial fibrillation of 76% compared to 26% in low-risk patients (P < 0.0001). The selective approach, i.e. treatment of high-risk patients only reduced the incidence of atrial fibrillation from 76% to 50% (P = 0.0295) compared to a reduction from 44% to 26% (P = 0.0065) when all patients were treated. More importantly, with the non-selective approach 100% of patients were exposed to the possible side-effects of sotalol and costs compared to 24% only with the selective approach (P < 0.0001). CONCLUSIONS: Thus, a selective approach based on a clinical risk prediction algorithm should improve the cost-effectiveness and safety of low-dose sotalol in the prevention of atrial fibrillation after coronary bypass surgery.

Studies of Muc-1 mucin expression and polarity in the mouse mammary gland demonstrate developmental regulation of Muc-1 glycosylation and establish the hormonal basis for mRNA expression.

Muc-1 is a major mucin glycoprotein expressed on the surface of mammary epithelial cells. It has attracted considerable attention as it is expressed in an aberrant form on many breast tumor cells. Here we describe studies using a recently obtained cDNA probe of Muc-1 expression during lactogenic development in the mouse. Northern blot analysis demonstrated that Muc-1 is expressed at all stages of lactogenic development but its levels are increased very significantly during mid-pregnancy and into lactation. The basis of this was examined using CID-9 mammary epithelial cell cultures. It was found that in the presence of insulin Muc-1 mRNA levels were increased by both hydrocortisone and prolactin, with the combination of the three hormones supporting maximum expression. Muc-1 mRNA levels were also modulated by culturing cells on a basement-membrane-like extracellular matrix that promoted mRNA levels 5- to 10-fold above levels in cells cultured on plastic tissue culture dishes. Immunocytochemical studies using monoclonal antibodies to carbohydrate epitopes on Muc-1 demonstrated that while Muc-1 was found at all developmental stages, it became increasingly sialylated during the course of pregnancy and into lactation. Additionally, we found that while Muc-1 is tightly polarized to the apical surface of the epithelium of lactating and pregnant mice it exhibited a less-polarized distribution on a small proportion of ductal cells in virgin mice. We conclude that the expression of Muc-1 is regulated at several different levels and by a number of different factors. We speculate that this may reflect different functional roles for Muc-1 at different stages of mammary development.

Airbag protection versus compartment intrusion effect determines the pattern of injuries in multiple trauma motor vehicle crashes.

OBJECTIVE: A prospective study of the interaction between airbag (AB) and seat-belt (Bt) protection versus vehicular compartment (VC) intrusion effects on injury patterns in motor vehicle crash (MVC) trauma patients. METHODS: Two hundred MVC patients, nonejected drivers or front seat passengers with multiple trauma or severe lower extremity (LE) trauma admitted to two Level I trauma centers. RESULTS: In frontal crashes, airbags (AB) more than Bt reduced Glasgow Coma Scale severity in brain injury, face fracture, shock, and the need for MVC extrication (all p < 0.05). Frontal AB also had a protective effect on LE fractures (41% vs. 66%, p < 0.01), but had no significant protective effect on pelvic fractures. When AB protection was present, it prevented brain and face fracture injuries caused by impact contacts and reduced the incidence of these injuries resulting from VC intrusions (p < 0.05). Thoracoabdominal injuries resulting from steering wheel intrusion showed AB protection against intrusions of twice the magnitude of those seen in non-AB vehicles (p < 0.05). In frontal MVCs, AB reduced LE fracture contact injuries but did not prevent LE fractures resulting from intrusions of instrument panel, toepan, or floor pedal structures. In lateral MVCs, Bt did not protect against brain, face, thorax, or pelvic injuries. CONCLUSIONS: Safety measures beyond frontal airbags must address frontal crash LE injuries induced by steering wheel, instrument panel, and toepan passenger compartment structure intrusions. Lateral crash injuries may profit from side AB supplemental restraint protection.