Faculty Bibliography

Transforming growth factor beta 1 (TGF-beta 1), a homodimeric polypeptide (Mr 25,000), derives from inflammatory cells and acts as a chemoattractant for monocytes and fibroblasts. We report here that TGF-beta 1 is also the most potent chemoattractant yet described for human peripheral blood neutrophils. Recombinant TGF-beta 1 elicited dose-dependent directed migration of neutrophils under agarose that was inhibited in the presence of a neutralizing antibody to TGF-beta 1. Maximal chemotaxis was evoked by TGF-beta 1 at femtomolar concentrations, whereas conventional chemoattractants act at nanomolar concentrations: on a molar basis, TGF-beta 1 was 150,000 times more potent than fMet-Leu-Phe. In contrast, TGF-beta 1 provoked neither exocytosis nor the production of superoxide by neutrophils. We further analyzed the mechanism by which TGF-beta 1 elicits chemotaxis (GTPase activity, [Ca2+], and actin polymerization). In contrast to the conventional chemoattractant fMet-Leu-Phe, TGF-beta neither activated classic heterotrimeric guanine nucleotide-binding proteins nor provoked global mobilization of intracellular Ca2+. Chemoattraction by both fMet-Leu-Phe and TGF-beta 1 was inhibited by cycloheximide and actinomycin D. Moreover, chemotaxis in response to TGF-beta 1 was associated with the polymerization of actin. The selectivity and potency of TGF-beta 1 as a chemoattractant suggest that it elicits directed cell migration by means of a pathway that depends not on classic intracellular signals but on protein synthesis

Evidence has accumulated suggesting that the various isoforms of beta-type transforming growth factors (TGF-beta s) regulate important functions in the lung; however, the cellular source of these proteins is not well defined. Northern blot analysis of murine lung tissue demonstrates that mRNA transcripts for all three TGF-beta isoforms are found from birth through adulthood. Although the level of expression for each TGF-beta is variable during the first 2 wk post partum, all three isoforms are equal in the adult lung. Using in situ hybridization and immunohistochemical analysis, we have localized both mRNA and protein expression for all three isoforms of TGF-beta in the adult murine lung. At low magnification, immunohistochemical localization of TGF-beta proteins appears coincident in their pattern of expression with TGF-beta mRNAs in the large proximal conducting airways of the lung. However, on closer analysis, protein expression of all three TGF-beta isoforms is confined to the bronchiolar epithelium, while TGF-beta mRNA transcripts for each of the TGF-beta genes are found in smooth muscle cells and connective tissue fibroblasts lying subjacent to the epithelium. Although the levels of both TGF-beta mRNA and protein expression are high in the proximal bronchiolar tree, their signal intensities completely disappear as the terminal bronchioles progress to respiratory bronchioles. Additionally, in the lung vasculature, there is very high expression of all three TGF-beta mRNA transcripts in the smooth muscle cells of the large vessels. TGF-beta2 and TGF-beta but not TGF-beta1 proteins are expressed in these same smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoform-specific antibodies to TGF beta 1, TGF beta 2, and TGF beta 3 proteins were generated and have been used to examine the expression of these factors in the developing mouse embryo from 12.5-18.5 d post coitum (d.p.c.). These studies demonstrate the initial characterization of both TGF beta 2 and beta 3 in mammalian embryogenesis and are compared with TGF beta 1. Expression of one or all three TGF beta proteins was observed in many tissues, e.g., cartilage, bone, teeth, muscle, heart, blood vessels, lung, kidney, gut, liver, eye, ear, skin, and nervous tissue. Furthermore, all three TGF beta proteins demonstrated discrete cell-specific patterns of expression at various stages of development and the wide variety of tissues expressing TGF beta proteins represent all three primary embryonic germ layers. For example, specific localization of TGF beta 1 was observed in the lens fibers of the eye (ectoderm), TGF beta 2 in the cortex of the adrenal gland (mesoderm), and TGF beta 3 in the cochlear epithelium of the inner ear (endoderm). Compared to the expression of TGF beta mRNA transcripts in a given embryonic tissue, TGF beta proteins were frequently colocalized within the same cell type as the mRNA, but in some cases were observed to localize to different cells than the mRNA, thereby indicating that a complex pattern of transcription, translation, and secretion for TGF beta s 1-3 exists in the mouse embryo. This also indicates that TGF beta 1, beta 2, and beta 3 act through both paracrine and autocrine mechanisms during mammalian embryogenesis

Plasma fibronectin (Fn) is a constituent of cryoglobulins and has been shown to interact with immune complexes. In a previous report we demonstrated that Fn specifically bound to IgG immobilized on a solid matrix. To localize and biochemically characterize the sites on the Fn molecule involved in this interaction, Fn was enzymatically cleaved with subtilisin and subjected to IgG affinity chromatography. Three major polypeptide fragments of 16 kDa, 22 kDa, and a triplet of 26- to 29-kDa bound IgG. They were localized to three separate regions of the molecule by Western blot analysis using antisera to specific regions of the Fn molecule, by amino acid sequencing, and by their previously described heparin binding affinities. The 22-kDa fragment interacted with IgG under physiologic conditions and it is localized at the N-terminal of the Fn molecule. The 16-kDa and 26- to 29-kDa fragments bound to IgG under conditions of lower ionic strength; the former commences at residue 588, carboxyl-terminal to the collagen binding region and the latter begins at residue 1597, carboxyl-terminal to the cell binding domain. The interaction of Fn with Ig has significant implications in host defense and also in immune complex disease where basement membrane Fn may sequester immune complexes from the circulation

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK

The substrate specificity and direct catalytic activity of plasminogen activator (PA) was examined under conditions where its natural substrate, plasminogen, was missing or inhibited. PA, purified from cultures of transformed chicken fibroblasts, was incubated with purified preparations of potential substrates. The adhesive glycoprotein fibronectin, isolated from normal chicken fibroblast extracellular matrix, underwent limited but specific cleavage by PA in the absence of plasminogen. Analysis of the cleavage products by polyacrylamide gels under both reducing and nonreducing conditions indicated that PA-mediated cleavage occurred near the carboxyl terminus of fibronectin but on the amino-terminal side of the interchain disulfide bridge, thus disrupting the native dimeric fibronectin molecule. Under the identical conditions, chicken ovalbumin was not cleaved while the established substrate, chicken plasminogen, was extensively converted to plasmin. A monoclonal antibody, directed against avian PA and shown to inhibit plasminogen-free, cell-mediated matrix degradation, specifically inhibited the fibronectin cleavage. A human PA, urokinase, also cleaved fibronectin under plasminogen-free conditions yielding a limited number of high molecular weight cleavage products