Purification and initial characterization of a type beta transforming growth factor from human placenta.

Abstract

A polypeptide transforming growth factor (TGF) that induces anchorage-dependent rat kidney fibroblasts to grow in soft agar has been isolated from human placenta and purified to homogeneity. This polypeptide is classified as a type (3 TGF because it does not compete with epidermal growth factor (EGF) for membrane receptor sites but does require EGF for induction of anchorage-independent growth of indicator cells. Purification of this peptide was achieved by acid/ethanol extraction of the placenta, followed by gel filtration, cation exchange, and HPLC of the acid-soluble proteins. Homogeneity of the TGF-.3 from the final column was shown by its constant specific activity and amino acid composition across the peak of soft agar colony-forming activity and by its migration as a single band at Mr 23,000-25,000 on NaDodSO4/polyacrylamide gel electrophoresis. Under reducing conditions, the protein migrated on a gel as a single band at Mr 13,000. The purified placental TGF-(8 caused half-maximal growth stimulation of indicator cells in soft agar at 64-72 pg/ml (3 pM) in the presence of EGF at 2 ng/ml (0.34 nM).Transforming growth factors (TGFs) have been operationally defined as low molecular weight acid-stable polypeptides that induce nonneoplastic anchorage-dependent rat kidney cells to form colonies that grow in soft agar (1, 2). These TGFs have been shown to have important functional interactions with epidermal growth factor (EGF) and can be subdivided into at least two groups based on these interactions (3-5). One group, defined as type a, is able to compete with EGF for membrane receptor binding sites and includes sarcoma growth factor (1), a TGF purified from human melanoma cells (6), and EGF itself (5). The second group, type ,B, does not bind to the EGF receptor. Type /3 TGFs were originally isolated from nonneoplastic tissues (3) but have recently been shown to occur in neoplastic cells as well (4, 5, 7). Both TGF-a and TGF-P must be present to induce the growth of nonneoplastic cells in soft agar (8). The normal physiological functions of these potent growth factors are not yet known. It has been suggested that they may be involved in embryological development (9, 10), wound healing (11), and tissue repair (11). In this report, the presence of a TGF-,B in human placenta is documented and the total purification to homogeneity of a TGF-P derived from a human organ is described. MATERIALS AND METHODSSoft Agar Assay. Soft agar colony-forming activity was determined as described (2) except that the cells were stained (12) at the end of 1 wk in assay and the numbers and sizes of the colonies were determined using a Bausch and Lomb Omnicon image analysis system (3).Extraction. Normal term human placentas were frozen on dry ice within 30 min after delivery and stored at -60'C until used. Placentas were extracted as described (2) except that the homogenized tissue (600-1,000 g) was stirred in the acid/ethanol solution at room temperature for 2 to 3 hr prior to centrifugation. The resulting supernatant was adj...

Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease

“…TGF-β1 was first isolated from placenta 49 , but whether transplacental transfer occurs has not been determined. TGF-β-like activity has been detected in human and bovine colostrum 50 , and a TGF-β2-like protein has been isolated from bovine milk 51 .…”

Signal transduction pathways that contribute to myeloid differentiation

“…234 TGFb/SMAD signaling pathway TGFb is a potent negative regulator of hematopoiesis. 235 TGFb has several isoforms (TGFb1-TGFb5), [236][237][238] and binding of TGFb to its receptor (I, II or III) [239][240][241] activates the receptor serine/threonine kinase, which in turn activates several intracellular signaling pathways. The Smad protein family (Smad1-8) acts as mediators of TGFb signaling.…”

Characterization of transforming growth factor-beta (TGF-beta) receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein.

“…The activities of TGFf, notably in extracellular matrix remodeling, differentiation, and immunosuppression, point to potential roles for TGF-3 as a mediator of trophoblastic invasion of the uterus, trophoblast differentiation, and maintenance of pregnancy (Altman et al, 1990;Clark et al, 1990;1991;Lala and Graham, 1990;Tamada et al, 1990;Lala, 1991, 1992a,b;Morrish et al, 1991;Ritvos and Eramaa, 1991;Graham et al, 1992a,b;Lea et al, 1992). Placenta and amniotic fluid are both relatively rich sources of TGF-3 (Frolik et al, 1983;Altman et al, 1990), and the TGF-f2 isoform, in particular, has been implicated in placental function (Miller et al, 1989;Altman et al, 1990;Clark et al, 1990;1991;Lea et al, 1992 (Friedman and Skehan, 1979;Burres and Cass, 1987 Boston, MA, December, 1991(Lee et al, 1991Mitchell and O'Connor-McCourt, 1991b). …”