ALTERATIONS IN THE PHENOTYPIC EXPRESSION OF CULTURED ARTICULAR CHONDROCYTES BY A
PITUITARY GROWTH FACTOR (CGF)*
CHARLES J. MALEMUD DAVID P. NORBY
Department of Pathology Health Sciences Center
State University of New York at Stony Brook Stony Brook, New York
I. INTRODUCTION
Recent advances have implicated serum (Todaro et al., 1967;
Holley and Kiernan, 1968, 1971, 1974; Leffert, 1974; Fryklund et al., 1974; Turkington and Majumder, 1973) and organ-derived
(Armelin, 1973; Gospodarowicz, 1974; Jones and Gospodarowicz, 1974; Lim and Mitsunobu, 1974; Revoltella et al., 1974; Cohen and Carpenter, 1975) factors as playing major roles in the control of proliferation and phenotypic expression of cultured cells. Such factors have been studied in attempts to find serum fractions which can be used as substitutes for whole serum (Pierson and Temin, 1972; Dulak and Temin, 1973; Holley and Kiernan, 1974), to study the mechanisms by which these factors interact with the
•Abbreviations used: CGF, Chondrocyte Growth Factor; MSA, Mul- tiplication - Stimulating Activity; EGF, Epidermal Growth Factor;
FGF, Fibroblast Growth Factor; NIH-TSH, Thyrotropin; MEM, Minimal Essential Medium; DMEM, Dulbecco's Modified Eagle's Medium; BSA, Bovine serum albumin; DG-2-deoxy-D-glucose; 2-AIB, 2-aminoisobu- tyric acid; Td-thymidine.
481
482 CHARLES J. MALEMUD AND DAVID P. NORBY
cell (Revoltella et al., 1974; Rudland et al., 1974a) and most recently to investigate bovine pituitary and brain molecules which profoundly augment the growth of cells not necessarily de- rived from these organs (Gospodarowicz, 1974, 1975). One of these pituitary factors has been designated chondrocyte growth factor
(CGF) because of its relative selectivity for promoting the growth of cultured articular chondrocytes (Corvol et al., 1972). CGF has not been purified and is recognized as a contaminant of sever- al pituitary glycoprotein hormones.
II. METHODS AND MATERIALS
The majority of experiments performed with CGF have utilized NIH-TSH, lots B5, B6, and B7 (2.47-3.53 U/mg protein) and its ef- fectiveness in promoting growth has been tested primarily on rab- bit articular chondrocytes grown under monolayer conditions as previously described (Sokoloff et al., 1970; Green, 1971; Corvol et al., 1972). The concentration of TSH in these cultures has generally been 64-70 yg/ml. Recently we have tested various pre- cursor fractions of TSH for CGF activity. These fractions desig- nated LER-1853-B2 (a crude pituitary glycoprotein fraction) and LER-1874 (a fraction derived from CG-50 chromatography of LER- 1853-B2) generously supplied by Dr. L. E. Reichert, are quite ef- fective in promoting the growth of lapine chondrocytes; the growth response towards 1853-B2 being in the same range as NIH-TSH (e.g.
200-250% above control values) while that of LER-1874 was slightly higher (333%).
III. RESULTS AND DISCUSSION
The effects of CGF as NIH-TSH on cultured chondrocytes have been summarized in Table I. The role of CGF as a growth-promot- ing factor was first demonstrated by an increase in total DNA
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484 C H A R L E S J . M A L E M U D A N D D A V I D P . N O R B Y
content/culture flask of CGF-treated cells. CGF augmented DNA synthesis which was maximal at 48 hours after subculture of pri- mary cultures and declined thereafter. CGF also shortened the generation time from 16 to 10 hours. Three of the cell cycle phases. S, G 2 , and M were identical with respect to time in con- trol and CGF-treated cultures. The G-^ phase of CGF-treated chon- drocytes, however, was almost totally absent, thus accounting for the shortened generation time.
In addition to promoting the growth of chondrocytes, CGF had a negative effect on RNA, protein and sulfated glycosaminoglycan synthesis. Thus, the protein, RNA content and net synthesis of sulfated glycosaminoglycans was reduced in CGF-treated chondro- cytes. However, the profile of the glycosaminoglycan species common to rabbit articular cartilage and cultured chondrocytes
(Srivastava et al., 1974) was not altered. The per cent of 3 5S 04 incorporated into chondroitin 4 and 6 sulfates or doubly-sulfated chondroitin was similar in control and CGF-treated chondrocytes
(Table I). Although glucosamine incorporation was similarly re- duced, synthesis of hyaluronic acid was unaffected by CGF (Male- mud and Sokoloff, 1974). Thus, the reduction in glycosaminogly- can synthesis was specific for the sulfated types.
The effect of CGF on sulfated glycosaminoglycan synthesis as well as on proliferation was reversible. When chondrocytes were initially grown in CGF-containing medium and CGF was then with- drawn and replaced with control medium, increments in growth and
35
reduction in ~^S04 incorporation were lessened when CGF was with- drawn at earlier intervals in the culture period.
Collagen synthesis was also reduced by CGF. Chondrocytes were cultured in the presence of LER-1874 (70 yg/ml) for 5 days.
With the last medium change, the cells were labeled with 3H - glycine (0.5 yCi/ml) in MEM with Earle's Salts supplemented with sodium ascorbate (100 yg/ml) and 10% fetal bovine serum for 20 hours. The medium was dialyzed against 0.2 M NaCl for 24 hours and the retentate counted for radioactivity. The cell pellet was
PHENOTYPIC EXPRESSION OF CHONDROCYTES 485 TABLE II Effect of CGF (as LER-1874)
on 3H-glycine Incorporation by Articular Chondrocytes in Monolayer Culture
H-glycine incorporation dpm x 103/yg DNAa
DNA Cellular Medium Group (yg/flask) TCA-insol. TCA-sol. non-dialyzable
Control 24.2 20.7 ± 1.3 5.48 ± 0.20 3.26 ± 0.16 LER-1874
(70 yg/ml) 104.8 2.98 ± 0.12 1.15 ± 0.06 0.64 ± 0.03
aMean ± s.e, (6 determinations),
precipitated with 6% TCA and the soluble and insoluble fractions counted for radioactivity. The results of this experiment are shown in Table II. They indicate that the total ^H-glycine in- corporation into the medium and cell pellet compartments were re- duced in the presence of LER-1874. These results reflected the overall reduction in total protein synthesis in the presence of CGF, but further experiments (see below) indicated that the pro- portion of 3H-glycine incorporated into collagen as distinct from other cellular protein was also quantitatively reduced.
An analysis of the ratio of al to a2 collagen chains was car- ried out primarily on the cell pellet fraction, since 80-85% of the Ç-glycine incorporated into collagen in 20 hours appears in this fraction (Norby et al., 1977). After treatment of this frac- tion according to methods to be described elsewhere (Norby et al., 1977), collagen was separated from proteoglycan by DEAE-cellulose chromatography (Miller, 1971). The chain composition of the col- lagen was then analyzed by CM-cellulose chromatography (Miller, 1971). Absorbance was monitored at 280 nm and 6.5 ml fractions collected. Alternate fractions were counted for radioactivity.
Radioactivity appearing in fractions corresponding to carrier collagen peaks were pooled for each peak to give al/a2 ratios.
486 CHARLES J. MALEMUD AND DAVID P. NORBY
The total °H-glycine incorporated into the cell pellet frac- tion of collagen was reduced in CGF-treated cultures (Control 15.3 x 1 04 dpm/mg DNA; LER-1874, 0.35 x 1 04 dpm/mg DNA). The al/
a2 ratio of control cultures was 5.37, suggesting a mixture of Type I and Type II collagens (Norby et al., 1977),
while that of CGF-treated cultures was 1.4. The greatly reduced Ç-glycine incorporation into the collagen of CGF-treated cul- tures made it difficult to conclude whether LER-1874 induced an alteration in the al/a2 ratio. Unlike the control cultures, more
3H-glycine incorporation into collagen was found in the medium than in the cell pellet of the CGF-treated cells. When the medi- um of LER-1874 cultures was analyzed by a modification of the method described above, the al/a2 ratio was 2.96, suggesting the presence of a mixture of Type I and Type II collagens.
The above studies have suggested that in addition to affect- ing chondrocyte proliferation in a positive manner, CGF suppressed the phenotypic expression of protein, glycosaminoglycan and col- lagen synthesis. These results are quite unlike that reported for other growth-promoting agents such as MSA (Pierson and Temin, 1972; Dulak and Temin, 1973), EGF (Cohen and Carpenter, 1975;
Hollenberg and Cuatrecasas, 1975) and FGF (Gospodarowicz, 1974, 1975). These latter growth factors stimulate not only prolifera- tion, but also most synthetic cell processes. Thus, the likeli- hood existed that CGF exerted its pleiotypic effect (Hersko et al., 1971; Rudland et al., 1974b) by limiting the transport of precursors required for incorporation into synthetic processes.
Conversely, augmentation of growth might have resulted from in- creased transport of nutrients which by an unknown mechanism were shunted into the pathways directed towards growth.
Table I shows that the transport of 2-DG (a glucose analogue) and 2-AIB (an amino acid analogue) was reduced in the presence of
PHENOTYPIC EXPRESSION OF CHONDROCYTES 487
CGF (as NIH-TSH) . These measurements were made 66 hours after in- troducing CGF to the cells, An antecedent effect of CGF appeared to be a requirement for reduced 2-DG transport, inasmuch as treat- ment of control cultures with CGF only at the time of measuring uptake failed to have any effect on transport of 2-DG. Thus, the effect of CGF on reducing 2-DG and AIB transport may not be di- rectly involved in altering membrane transport, but may instead be involved in producing changes in the cell membrane which reduce the capacity of the CGF-treated cell to transport these small molecules. It should also be noted in this regard that CGF does not support the growth of chondrocytes in the absence of serum.
When cells were grown in BSA (3 mg/ml) or BSA + CGF (20 yg/ml), growth either did not occur or was not sustained. (Figure 1 ) . This suggested the possibility that CGF activated a serum compo- nent in order to augment growth.
488 CHARLES J. MALEMUD AND DAVID P. NORBY
FIGURE 1 Failure of cultured articular chondrocytes to pro- liferate in the absence of fetal bovine serum. 2A. Normal con- trol chondrocytes grown for 4 days in DMEM +10% FBS. 2B. Chon- drocytes grown in the absence of fetal bovine serum, with 3 mg/ml BSA as protein source. Cells have attached, with greatly reduced plating efficiency, and have not spread out. 2C. Chondrocytes grown in BSA, 3 mg/ml + CGF (20 \xg/ml) . Some cells have attached
PHENOTYPIC EXPRESSION OF CHONDROCYTES 489 ACKNOWLEDGMENT
We wish to thank the Hormone Distribution Officer of The National Institute of Arthritis, Metabolism and Digestive Dis- eases for supplying NIH-TSH and Dr. Leo E. Reichert, Emory Uni- versity School of Medicine for the LER-1853-B2 and 1874 fractions.
Supported by a grant from The National Institute of Arthritis, Metabolism and Digestive Diseases CAM 17258-01).
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