DEVELOPMENTAL
BIOLOGY
Transforming
143.303-308 (19%)
Growth Factor (3 Type 1 Binds to Collagen IV of Basement Membrane Matrix: Implications for Development VISHWASM.PARALKAR,SLOBODANVUKICEVIC,AND
A.H.
REDDI'
The interaction of transforming growth factor p (TGF 0) with extracellular matrix macromolecules was examined by using radiolabeled TGF B and various matrix macromolecules immobilized on nitrocellulose. TGF @bound to collagen IV with greater affinity than to other extracellular matrix macromolecules tested. Neither laminin nor fibronectin, both of which bind type IV collagen, interfered with the binding of TGF 13to type IV collagen. TGF &competed effectively with TGF & for binding to type IV collagen. The biological effect of TGF /j was tested by an assay based on inhibition of proliferation of an osteoblast cell line, MC3T3-El. The results demonstrated that the effect of TGF & was sustained when cells were grown on type IV collagen compared to cells grown on laminin, collagen type I, and plastic. These results demonstrate that extracellular matrix components may function as an affinity matrix for binding and immobilizing soluble growth and differentiation factors. In view of the demonstrated role of basement membranes in development the present results imply an important function for transforming growth factor $ hound to collagen IV in local regulation of cell proliferation and differentiation. ij 1981 Academic Press, Inc.
brane, and this finding tion.
INTRODlJCTION
Transforming growth factor fi (TGF 0) is an important mediator of numerous cellular and physiological processes (Sporn ef al., 1987; Ignotz and Massague, 1986). Although several isoforms of this factor exist, considerable work has been done on TGF p, and TGF & (Cheifetz et al., 198’7; Seyedin et al., 1987). TGF fi stimulates the gene expression of the extracellular matrix macromolecules fibronectin and type I collagen (Roberts et al., 1986; Bassols and Massague, 1988) and also regulates the metabolism of proteoglycans (Morales and Roberts, 1988; Madri et al., 1988). Extracellular matrix components in turn modulate the effect of TGF p on cells in vifro (Davis, 1988). There is a growing realization of the importance of extracellular matrix in cell differentiation and morphogenesis (Hay, 1981; Kleinman ef al., 1981; Reddi, 1984, 1985). Extracellular matrix components are involved in the attachment, proliferation, and differentiation of a variety of cells i7r ,uitro. In view of the known effects of TGF fi on extracellular matrix we investigated the affinity of this multifunctional regulator for matrix components. During the course of studies on the interaction of TGF p with other extracellular matrix macromolecules we found that transforming growth factor & (TGF &) bound avidly to type IV collagen, a major component of the extracellular matrix of basement mem-
1 To whom
correspondence
should
MATERIALS
is the focus of this communica-
AND
METHODS
Materials. Type IV collagen and laminin were purified from EHS sarcoma, a transplantable mouse tumor, and were found to be homogeneous by SDS-gel electrophoresis (Kleinman et al., 1982). Type I collagen was purified from an acid extract of rat tail tendon (Linsenmayer ef al., 1978). TGF p, platelet-derived growth factor (PDGF), epidermal growth factor, and insulin-like growth factor 1 were from R and D Systems, Minneapolis. Chondroitin sulfate was purchased from Seikagaku Kogyo Co., Japan, and heparin was from Fisher Scientific. Heparan sulfate, dermatan sulfate, and hyaluronic acid were from Calbiochem. Chloramine-T and bovine serum albumin (BSA) were from Sigma. Collagen types II and IX were gifts of Dr. Daniel Herbage. Iodimtion of TGF 8. TGF fil was iodinated according to the chloramine-T method as described (Frolick et al., 1984). Briefly, one pg of TGF p was iodinated with 300 PCi of NalZ51. Iodinated TGF fi was separated from free NaI by gel filtration through a G-25 column (Pharmacia) and the biological act,ivity was demonstrated by receptor binding to mink lung epithelial cells (MVlLU). Specific activity of labeled TGF p, was 100 pCi/pg. Iodinated material contained greater than 96% trichloroacetic acid precipitable material and was stored at -70°C in 0.1% BSA.
be addressed. 303
001%1606/91 Copyright Ail rights
$3.00
Cc ICI!)1 hy Academic Press. Inc. of rrpro
BIOLOGY
Transforming
143.303-308 (19%)
Growth Factor (3 Type 1 Binds to Collagen IV of Basement Membrane Matrix: Implications for Development VISHWASM.PARALKAR,SLOBODANVUKICEVIC,AND
A.H.
REDDI'
The interaction of transforming growth factor p (TGF 0) with extracellular matrix macromolecules was examined by using radiolabeled TGF B and various matrix macromolecules immobilized on nitrocellulose. TGF @bound to collagen IV with greater affinity than to other extracellular matrix macromolecules tested. Neither laminin nor fibronectin, both of which bind type IV collagen, interfered with the binding of TGF 13to type IV collagen. TGF &competed effectively with TGF & for binding to type IV collagen. The biological effect of TGF /j was tested by an assay based on inhibition of proliferation of an osteoblast cell line, MC3T3-El. The results demonstrated that the effect of TGF & was sustained when cells were grown on type IV collagen compared to cells grown on laminin, collagen type I, and plastic. These results demonstrate that extracellular matrix components may function as an affinity matrix for binding and immobilizing soluble growth and differentiation factors. In view of the demonstrated role of basement membranes in development the present results imply an important function for transforming growth factor $ hound to collagen IV in local regulation of cell proliferation and differentiation. ij 1981 Academic Press, Inc.
brane, and this finding tion.
INTRODlJCTION
Transforming growth factor fi (TGF 0) is an important mediator of numerous cellular and physiological processes (Sporn ef al., 1987; Ignotz and Massague, 1986). Although several isoforms of this factor exist, considerable work has been done on TGF p, and TGF & (Cheifetz et al., 198’7; Seyedin et al., 1987). TGF fi stimulates the gene expression of the extracellular matrix macromolecules fibronectin and type I collagen (Roberts et al., 1986; Bassols and Massague, 1988) and also regulates the metabolism of proteoglycans (Morales and Roberts, 1988; Madri et al., 1988). Extracellular matrix components in turn modulate the effect of TGF p on cells in vifro (Davis, 1988). There is a growing realization of the importance of extracellular matrix in cell differentiation and morphogenesis (Hay, 1981; Kleinman ef al., 1981; Reddi, 1984, 1985). Extracellular matrix components are involved in the attachment, proliferation, and differentiation of a variety of cells i7r ,uitro. In view of the known effects of TGF fi on extracellular matrix we investigated the affinity of this multifunctional regulator for matrix components. During the course of studies on the interaction of TGF p with other extracellular matrix macromolecules we found that transforming growth factor & (TGF &) bound avidly to type IV collagen, a major component of the extracellular matrix of basement mem-
1 To whom
correspondence
should
MATERIALS
is the focus of this communica-
AND
METHODS
Materials. Type IV collagen and laminin were purified from EHS sarcoma, a transplantable mouse tumor, and were found to be homogeneous by SDS-gel electrophoresis (Kleinman et al., 1982). Type I collagen was purified from an acid extract of rat tail tendon (Linsenmayer ef al., 1978). TGF p, platelet-derived growth factor (PDGF), epidermal growth factor, and insulin-like growth factor 1 were from R and D Systems, Minneapolis. Chondroitin sulfate was purchased from Seikagaku Kogyo Co., Japan, and heparin was from Fisher Scientific. Heparan sulfate, dermatan sulfate, and hyaluronic acid were from Calbiochem. Chloramine-T and bovine serum albumin (BSA) were from Sigma. Collagen types II and IX were gifts of Dr. Daniel Herbage. Iodimtion of TGF 8. TGF fil was iodinated according to the chloramine-T method as described (Frolick et al., 1984). Briefly, one pg of TGF p was iodinated with 300 PCi of NalZ51. Iodinated TGF fi was separated from free NaI by gel filtration through a G-25 column (Pharmacia) and the biological act,ivity was demonstrated by receptor binding to mink lung epithelial cells (MVlLU). Specific activity of labeled TGF p, was 100 pCi/pg. Iodinated material contained greater than 96% trichloroacetic acid precipitable material and was stored at -70°C in 0.1% BSA.
be addressed. 303
001%1606/91 Copyright Ail rights
$3.00
Cc ICI!)1 hy Academic Press. Inc. of rrpro
