Gary R. Grotendorst

Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media☆

Human retinal pigment epithelial cells at confluence were used to condition serum-free Dulbeccos modified Eagles medium. Conditioned media were exhaustively dialyzed against 0.5 N acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from goat antiserum directed against human platelet-derived growth factor. Native platelet-derived growth factor was resolved as a band with Mr of 30 kDa under non-reducing conditions, while bands with Mr of 36-38 kDa and 18.5 kDa were resolved from retinal pigment epithelial cell-conditioned media. Acid extracts of retinal pigment epithelial cells also contained bands at 36-38 kDa and media conditioned for 48 hr exhibited much denser bands than media conditioned for 24 hr. No bands were detected when non-immune goat IgG fractions were substituted for primary antibody and when conditioned media were prepared from several human fibroblast lines in the same manner as those prepared from retinal pigment epithelial cells, no detectable bands or only a faint shadow at 36 kDa were seen. Retinal pigment epithelial cell-conditioned media prepared in the presence of [35S]methionine were loaded on an anti-platelet-derived growth factor IgG affinity column, eluted, and subjected to SDS-polyacrylamide gel electrophoresis. Bands with Mr slightly less than 36 kDa and 18 kDa were visualized by autoradiography, demonstrating that the platelet-derived growth factor-like proteins in retinal pigment epithelial cell-conditioned media are newly synthesized. Two fractions eluted from the column also markedly stimulated fibroblast chemotaxis and incorporation of [3H]thymidine, both of which were neutralized by soluble anti-platelet-derived growth factor IgG. These data suggest that retinal pigment epithelial cells in culture produce platelet-derived growth factor-like proteins and secrete them into their media where they are capable of stimulating fibroblast chemotaxis and proliferation.

Connective tissue growth factor

Connective tissue growth factor (CTGF) is secreted by human umbilical vein endothelial cells and has a biological activity related to that of platelet-derived growth factor (PDGF). Human umbilical vein endothelial cells have been previously reported to secrete PDGF-related factors into the media. DiCorleto et at. reported that endothelial cells secrete factors that are chemotactic and mitogenic for connective tissue cells and around 30% of this activity was neutralized by antihuman PDGF antibodies (1). Collins et at. reported that human umbilical vein endothelial cells express both the A and B chain genes of PDGF (2, 3). Thus the mitoattractant activity found in human umbilical vein endothelial cells was thought to be due to PDGF gene products. However, the exact nature of this protein was uncertain. We tried to determine the properties of the PDGF-related molecules secreted by human umbilical vein endothelial cells and found a new peptide distinct from PDGF (4). Confluent human umbilical vein endothelial cells were incubated in serum free media for 6 and 48 hours. Then conditioned media was recovered and analyzed by Western blotting which was probed with anti-PDGF antibody. We detected 36-38kd molecules which were bigger than PDGF B chain dimers. Neither anti amino terminal A chain nor anticarboxy terminal B chain antibody picked up this band, indicating that this peptide does not contain PDGF A or B chain peptides. In reduced conditions, the band appeared at 38kd, indicating that this peptide is a monomer. We termed this protein CTGF. We purified CTGF from the conditioned media using an affinity column made with the anti-PDGF antibody and an Affigel 10 support. We performed chemotactic and mitogenic assays using NIH3T3 cells. These data show that CTGF has biological activities affecting these cells, as do PDGF isoforms.

Alteration of cyclic nucleotide levels in phorbol 12-myristate 13-acetate treated myoblasts

Summary The tumor promoter phorbol 12-myristate 13-acetate alters the levels of both cGMP and cAMP in chick myoblasts. There is a transient 7-fold increase in cellular cGMP 40–60 sec after promoter addition. The changes in cAMP are slower with a 50% decrease at 30 min followed by a 3.5-fold increase at 3 h. The relationship between these cyclic nucleotide changes and the previously reported stimulation of hexose transport by the promoter [Fed. Proc. 37, 1817 (1978)] was investigated. Addition of 8-bromo cGMP, 8-bromo cAMP or phosphodiesterase inhibitors had no effect on hexose transport while LiCl (an inhibitor of adenyl cyclase) was stimulatory. The tumor promoter and LiCl, however, appear to stimulate hexose transport by different mechanisms.

Binding of phorbol-12-myristate-13-acetate to cultured myoblasts☆

The tumor promoter phorbol 12-myristate 13-acetate (PMA) binds in a rapid (maximal at 30 min), reversible, and non-saturable manner to cultured embryonic chick myoblasts. Serum was not required for binding although it promoted the release of PMA from the cells. This appears to be due to PMA binding to serum proteins. At any concentration tested, approx. 10% of the total PMA was bound at 5 min and 30% was bound at 30 min. It is estimated that a PMA concentration in the cell membrane of only 1 molecule of PMA/5000 molecules of membrane lipid elicits maximal biological responses in these cells.