Vitamin A inhibits chondrogenesis but not myogenesis

Preliminary tain cell functions [ 191, and it is tempting to consider this interrelationship operative in modulating cellular differentiation. Thus, it is conceivable that certain embryonic cells develop a calcitonin-sensitive cyclase system to modulate CAMP and calcium levels, and in this manner regulate specific cellular processes during development.

I. Pierce, G B, Stevens, L C & Nakane, P K, J natl cancer inst 39 (1967) 755. Martin, G R, Cell 5 i1975) 229. Bonner. J T, Proc natl acad sci US 65 (1970) I IO. Zalin, R J & Montague, W. Cell 2 (1974) 103. Prasad, K N & Hsie, A W, Nature new biol 233 (1971) 141. 6. Marcelo, C L, Exp cell res 120 (1979) 201 7. Bernstine, E G, Hooper, M L, Grandchamp, S & Eohrussi, B. Proc natl acad sci US 70 (1973) 3899. 8. &gal. S & Khoury, G. Proc natl acad sci ‘US 76 7 i: 4. 5.

(1979)

5611.

Anderson, W B, Gallo, M & Pastan, I, J biol them 249

10.

(1974)

469

Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827/801110469-OSSO?.OO/O

Vitamin A inhibits chondrogenesis but not myogenesis MAURIZIO MOLINARO logia Roma. Virali,

PACIFICI,’ GIULIO COSSU. MARIO and FRANC0 TAT0,2 Istitufo di Isto-

ed Embriologia and ‘Laboratorio Istittcto Superiore

generale,

Universitci di Roma, di Malattie Batteriche e di Sanita. Roma, Italy

Limb bud cells were isolated from HH stage 22-23 chick embryos and were grown as a ‘spot culture’ in in vitro conditions which support their differentiation into chondrocytes and myotubes. By day 4 of culture, numerous chondrocyte nodules developed and were scattered mainly in the very centre of the cell spot. In contrast, multinucleated myotubes formed at both the centre and the periphery of the cell spot. Treatment with vitamin A starting on day 1, inhibited chondrogenesis in these cultures, and by day 4-6 chondrocyte nodules could not be detected histologically. In contrast, no dose of vitamin A tested was effective in suppressing the development of multinucleated myotubes. These data show that vitamin A selectively inhibits chondrogenesis but not myogenesis in limb bud cell cultures.

Summary.

References

9.

notes

7041.

Harper, J F & Brooker, G J. Cyclic nucleotide res l(l975)

107.

Miller, Z, Lovelace, E, Gallo, M & Pastan, I, Science 190 (1975) 1213. 12. Bohlen, P, Stein, S, Dairman, W & Udenfriend, S, Arch biochem biophys 155 (1973) 213. 13. Perkins, J P, Adv cyclic nucleotide res 3 (1973) 1. 14. Marx, S J, Wooddard, C J & Aurbach, G D, Science 178 (1972) 999. 15. Johannes, N, Heersche, M, Marcus, R & Aurbath, G D, Endocrinology 94 (1974) 241. 16. Fischer, J A. Hunziker. W & Moran. J, Molecular endocrinology (ed I MacIntyre & M Szelke) p. 193. Elsevier, Amsterdam (1977). 17. Munson. P L. Handbook of ohvsiologv (ed G D Aurbach) vol: 7, p. 443. A&&an Physiological Science. Washinaton. D.C. (1976). 18. Ardaillou, R. Nephron 15 (1975) ZSO. 19. Berridge. M J. Adv cyclic nucleotide res 6 (1975) 1. II.

Received February 28, 1980 Revised version received May 27, 1980 Accepted June 5, 1980

Hypervitaminosis A has long been known to exert teratogenic effects on developing embryos. Excessive dietary intake of vitamin A by pregnant animals produces several congenital malformations in the fetuses [l-2]. More recently, by injecting or feeding pregnant animals with a single massive dose of vitamin A it has been shown that characteristic limb malformations, such as micromelia or phocomelia, emerge dependent upon the day of pregnancy at which vitamin A is administered [3-4]. These limb defects are not accompained by other major abnormal body structures [4]. It has been suggested that the cellular target of the teratogenic action of vitamin A is represented by the centrally located chondrogenic cells in the developing limb, which in turn fail to organize normal cartilaginous models of the limb skeletal bone [4]. ’ To whom offprint requests should be addressed: Department of Anatomy School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Printed

m Sweden

Exp Cell Res 129 (19801

conditions \+,hich support their final differentiation into chondroc),tes [>I. In thehe e\perimental conditions. the chronic e\posure of these cells to hypervitamino\is A blocks chondrogenesis [5]. As also shown here. proper culture conditions can promote the emergence of both chondrogenic and myogenic cells in limb bud cell cultures. which. respectively. lead to the development of metachromatically stainable chondrocyte nodules and crossstriated. multinucleated myotubes [C-7]. In these experimental conditions. tire tested whether hypervitaminosis .4 would have similar or differential effects on the emel-gence of these two mesodermal. phenotypically distinct cell types. Our results demonstrate that vitamin A selectively blocks chondrogenesis but not myogenesis in developing limb bud cells in culture.

I. Micrographs of toluidine blue-stained da) Jlimb bud cell cultures. Cells were beaded on I(I) uncoated; tD. c) collagen-coated dishes. Note the pre+ ence of both chondrocyte nodules ((‘)I) and myotubeh (nr.v) in (il. h) control cultures. whereas only myotubes are present in ((,I vitamin A-treated culture. x 125. Fi,q

The study of the teratogenic effects of vitamin A on limb development has recently been re-approached by isolating and growing embryonic limb bud cells in culture

HH stage 22-23 chick embryo limb bud5 [8] wel-e isolated and dissociated into single-cell suspension by treatment with trypsin-EDTA solution for 30 min. Cells were concentrated by centrifugation. resuspended in complete medium 1854 MEM+lO”r fetal calf berum (FCS)+3% embryo extract) and adjusted to 20-25~ IO’ cells/ml. IO-20 ul aliauots were then plated down as ‘spots’ on uncoated or’collagen-coated 35 mm dishes [Sl. I-1 h later 2 ml of complete medium were added. Medium was changed evkry other day. Cultures were fixed on day +6 with IO% formalin in BSS for I h. stained with I? toluidine blue aqueous solution for I min. destained with 95q ethanol and analysed microscopically. Alternatively. the, were stained with Alcian blue 8GX. pH 1.0 [IO]. Homogeneous populations ?f skeletal myogenic cells were obtained from I I-day-old chick embryo breast muscles by selective removal of contaminating fibroblasts [I Il. 7.5~ lOi cells were plated onto 60 mm collagen-coated dishes and grown in MEM containing IO?: FCS. Pure populations of differentiated embryonic chondrocytes were prepared and grown as detailed elsewhere 191. All &idia were from Gibco: retinoic acid and retinol acetate from Sigma: retinol was a gift of Dr L. M. De Luca. NCI. NIH.

Krsrrlts Limb bud cells plated as a ‘spot’ on uncoated plastic dishes and grown in the cul-

Preliminary

Table 1. Dose-dependent metachromnticnlly-stainable tinoids Retmoid

inhibition oj nodules by re-

concentration

(pg/ml)

Type of substratum

0

0.1

0.5

I.0

2.5

Plastic Collagen

++++ ++

++ +-

f-

-

-

Limb coated added units: spot;

bud cells were grown on uncoated or collagendishes and stained on day 4. Retinoids were on day 1. Results are expressed in arbitrary ++++. over 200 metachromatic nodules per -, absence of nodules.

ture conditions described here adhered to the substratum within 30-40 min. and started to proliferate. During the initial 2448 h, the area just outside the periphery of the original spot was invaded by cells of indistinct phenotype. Clearly distinguishable multinucleated myotubes appeared on top of these peripheral cells by the end of day 2. During the following 24-48 h, numerous myotubes emerged also in the centre of the cell colony (fig. 1 a). Unequivocal chondrocyte nodules, on the contrary, became recognizable only between day 2 and day 3, and were scattered exclusively within the limits of the original cell spot. These nodules then grew in size and in number; by the end of day 4 they occupied most of the central area of the spot and could be clearly stained metachromatically or with Alcian blue (fig. 1 a). Morphologically differentiated chondrocyte nodules have been shown to synthesize cartilage-specific products, such as type IV sulphated proteoglycans and type II collagen chains [9, 12, 131. When limb bud cells were plated on collagen-coated dishes, extensive myogenesis occurred even during the initial 2 days of culture, and by day 4 numerous myotubes occupied both the periphery and the centre

notes

471

of the cell spot (fig. 1 b). Fewer, though unequivocal chondrocyte nodules emerged at the centre of the cell colony (fig. lb). In a successive set of experiments, limb bud cell cultures were treated with various amounts of retinoids, which were added to the growth medium at 24 h of culture. Table 1 summarizes the dose-dependent effects of retinoic acid (vitamin A acid) on the development of metachromatically stainable chondrocyte nodules. In brief, 0.5 pg/ml of retinoic acid were effective in suppressing chondrogenesis in limb bud cell cultures prepared on uncoated dishes. On the other hand, as little as 0.1 pg/ml of retinoic acid suppressed chondrogenesis in cultures grown on collagen-coated dishes (fig. 1 c). Similar results were obtained with retinol and retinol acetate. Conversely, any dose of vitamin A tested was ineffective in inhibiting myogenesis. As shown in fig. 1c, while no chondrocyte nodules appeared, multinucleated myotubes developed at the centre and the periphery of vitamin A-treated cultures. Since, as shown above, myogenesis was promoted on collagen-coated dishes, the lack of vitamin A effects on myogenesis was more evident at a microscopic level in cultures prepared on this substratum. Myotubes in both control and vitamin A-treated cultures could be stained with antibodies to adult skeletal muscle myosin, localized with a direct immunofluorescent iechnique; by day 6 of culture, myotubes exhibited clear areas of cross-striation (M. Pacifici, J. Croop & H. Holtzer. Unpublished). On the one hand, the differential responses of chondrogenic and myogenic cells reported above might be a direct consequence of a differential sensitivity of these two distinct cell types to hypervitaminosis A. On the other hand, the observed responses might instead be mediated, Exp Cell

Res 129 11980)

Fig. and ture.

.?. Micrographs of cultured skeletal muscle cells vertebral cartilage chondrocytes. day 4-C of cul(n) Control muscle cells: (h) 2.5 pg/ml-treated

modulated or interfered with by the other cell types present in such high cell-density cultures, and therefore exert an indirect effect. In an effort to clarify these points, the following two sets of experiments were performed. We studied the effects of vitamin A on populations of skeletal myogenic cells isolated from 11-day-old chick embryo breast muscles. As known. these cells are able to complete their differentiation in culture: in standard conditions they grow and can develop at rather low density. We tested retinoic acid and retinol acetate doses up to 10 pg/ml and retinol doses of 2-3 pg/ml, all of which appeared ineffective in suppressing myogenesis, both qualitatively and Exp Cell

Res 129 (19801

muscle treated

cells; ((.I control chondrocytes.

ihondroc)te\: x 13.

~JI Il.5

@g/m-

quantitatively (fig. 2 CI, 6). Both vitamin Atreated and control cultures contained a similar number of cells, and more than 70 9: of the total nuclei fused into myotubes by day 4. Higher doses of retinoids had toxic effects. We also analysed the effects of vitamin A on embryonic, fully developed chondrocytes grown in culture conditions identical with those used for skeletal muscle cells. Within Z-4 days of treatment with 0.2-0.5 pg/ml of retinoic acid or retinol acetate. chondrocytes lost their typical epithelioid appearance, could not any longer be stained metachromatically and transformed into flat, irregularly shaped cells (fig. 3c, d). As reconfirmed at this laboratory. concurrent

Preliminnt-y with their morphological transformation the chondrocytes lost the ability to synthesize their specific products [ 14-161. Discussion The mechanisms by which vitamin A selectively inhibits chondrogenesis but not myogenesis in developing limb bud cell cultures, are not clear at the moment. Vitamin A treatment has been reported to partially inhibit cell proliferation in high cell density cultures of limb bud cells [17]. The selective inhibition of chondrogenesis reported here may suggest that vitamin A treatment selectively inhibits cell proliferation of precursor chondrogenic cells without affecting that of myogenic cells. However, though direct evidence of this is still lacking, this interpretation may also explain other similar experimental observations. as also discussed in ref. [18]. In addition, as also reported here, vitamin A alters the expression of the specific characteristics of mature duplicating chondrocytes in culture [ 15-161, and has similar effects on cultured rudiments in which there is practically no mitotic activity [ 191. Therefore, it remains to be clarified whether vitamin A treatment (1) prevents developing limb bud chondrogenic cells from differentiating into chondrocytes; (2) blocks the expression of their differentiated phenotype without affecting the progression through the final stages of their development; or (3) selectively blocks chondrogenic cell proliferation. Recent findings have shown that treatment with vitamin A induces accumulation of sizeable amounts of cellular frbronectin [20] as a cell surface component on phenotypically-altered mature chondrocytes and limb bud cells in culture [5, 211. Moreover, the addition of fibronectin to growth medium has been shown to transform ma31-801806

tlotes

473

ture chondrocytes into altered, fibroblastlike cells [22]. These findings could lead one to believe that one possible mechanism of action of vitamin A consists in the induction of sizeable amounts of cell surfaceassociated fibronectin, which in turn may alter the phenotype of vitamin A-treated cells. However, it has also been shown that (1) fibronectin is present but decreases during differentiation of L6-myogenic cells; and (2) addition of fibronectin to the growth medium blocks cell fusion of L6-myogenic cells [23-251. It is clear. therefore, that at this stage of our knowledge the vitamin A-induced accumulation of cell surface fibronectin does not represent a satisfactory explanation of the differential effects of hypervitamin A on myogenesis and chondrogenesis in cultured limb bud cells reported here. Vitamin A has been shown to be involved in normal glycosylation reactions during glycoprotein biosynthesis in a variety of cell types [26]. It has been proposed that the effects of vitamin A excess or deficiency are exerted by alteration in the glycosylation pathways of glycoproteins. As an alternative explanation of our results, this would suggest that qualitatively different mechanisms of protein glycosylation exist in differentiating chondrogenic and myogenic cells, differentially affected by excess vitamin A. Clearly our data demonstrate that the biological effects of vitamin A are variegated and strictly depend upon the specific genotypic program of the responding cell type. Our observations could be relevant to the conflicting proposal for retinoids as promoting or antitumoral agents for ectodermal, as well as mesodermal cells in vivo or in vitro [27-291, in that genetically distinct cell types might differentially respond to hypervitamin A treatment. E-rp Cd

Res 129 11980~

This work 79.01938.01

was supported and NATO grant

by no.

CNR Ih20.

go-ant

no.

I. Cohlan. S Q. Science I I7 I 19%) 535. 2. Giroud. A & Martinet, M. Arch franc _ nediat I? . (1955) 292. 3. Murakami. U & Kameyama. Y. .Arch rnvironhealth IO t 1965) 732. 4. Kochhar. D M. ‘Teratology 7 t 1973) 289. 5. Lewis. A C. Pratt. R M. Pennypacker-. J P & Hassell. J R. Dev biol 63 ( 197X) 31. 6. &plan. A I, Exp cell res 62 t 1970) 331. 7. Dienstman. S R, Biehl. J. Holtzer. S & Holtzer. H. Dev biol 39 (197-11 X3. 8. Hamburger. V & Hamilton. H W. J morphol $8 (1951) 49. 0. Okayama. M. Pacifici. M & Holtzer. H. Proc natl acad sci US 73 (1976) 3223. IO. Yamada. K, Histochemie 23 t 1970) 13. I I. Zani. B. Cossu. G. Adamo. S & Molinaro. M. Differentiation IO t 1978) 95. 12. van der Mark. K & von der Mark. H, J cell biol 73 t 1977, 736. 13. Holtzer. H. Okayama, M. Biehl. J Cy: Holtzsr. S, Experientia 34 t 1978) 781. IJ. Chacko. S. .4bbott. J. Holtzer. S & Holtzer. H. J exp med 130 t 1969) 417. IS. Solurch. M 2s Meier. S. Calcif tissue re\ I3 t 1973) 131. 16. Shapiro. S S & Poon. J P. .4rch biochem biophyb I71 t 1976) 73. J R. Pennypacker. J P& Lewis. .A C. Exp 17. Hassell. cell res I II t 1978) 309. H. Stem cells and tissue homeostasib. pp. IX. Holtzer. l-28. Cambridge University Press. Cambridge (197X). 19. Fell. H B & Dingle. J T. Biochem j X7 t 1963) 303. 20. Hynes. R 0 & Humphrey. K C. J cell biol 62 t 1971) 338. 21 Hassell. J R, Pennypacker. J P, Kleinman. H K. Pratt. R M & Yamada. K M, Cell I7 t 1979) 821. 22. West. C M. Lanza. B. Rosenbloom. I. Lowe. M. Holtzer. H & Avdalovic. N. Cell I7 t 1979) 491. ‘3 Chen. LB. Murray, A, Segal. R N. Bushnell. A & Walbh. M L. Cell I4 t 1978) 377. 24, Furcht. L T. Mosher, D F & Wendelskhafer-Crabb. G. Cell I3 t 197X) 263. IS Podleski. T R. Greenberg. 1. Schlessinger. J &: Yamada. K M, Exp cell res 122 (19791 317. L M et al.. Pure & appl them 51 (19791 26. De Luca. 581. 27. Hogan. B. Nature 277 (1979) 261. 28. Todaro. G J. De Larco. J F Br Sporn. M B. Nature 276 t 1978, 272. 29. Levine. L 8: Ohuchi. K. Natut-e 276 t 1978,271. Received Revised Accepted

March version June

7. 1980 received 25. 1980

June

23.

19X0

Possible involvement of arachidonic acid in the initiation of DNA synthesis by rat liver ceils

.S/r?>!nr~r~~. Calcium-deprived T5 IB rat liver cells initiated DNA synthesis within I h after addition of calcium. The possibility of this DNA-synthetic response having been mediated through arachidonic acid metabolism ti.e.. through an arachidonic acid cascade) is suggested by the fact\ that calcium is known to stimulate phospholipase activity which releases arachidonic acid from membrane phospholipids: that low concentrations t 10-!‘-lOmti mole/l) of arachidonic acid itself elicited the same DNA-synthetic response from the calcium-deprived cells as calcium; and that the stimulatory actions of calcium and arachidonic acid were both blocked b) the endoperoxide synthase inhibitor indomethacin.

Non-neoplastic cells in vitro and in vivo need both calcium ions and a transient increase in their CAMP content near the end of the GI phase of the growth-division cycle to initiate DNA synthesis [Z. 7. IO17, 19-261. Extensive experimentation in this and other laboratories has shown that lowering the extracellular calcium concentration or preventing the CAMP surge does not stop on-going DNA synthesis. but does stop the proliferative development of the cells in late Gl phase [2. 3. 7. IO. I I. cells revert 19-23. 251. These blocked sooner or later to an earlier prereplicative state [IO, I I. IS. 191. unless rescued by a timely addition of calcium [2. 3. 5, 10 20-251. calcium’s intracellular mediator cdmodulin [4] or a prostaglandin (e.g.. PGA or PGE,). anyone of which causes the cells to initiate DNA synthesis within I h [Z-5. 8. 13. 16, 17. 20. 21. 241. Since calcium and its mediator calmodulin might release arachidonic acid from membrane by stimulating phospholipases [6. 9, 761. and since prostaglandins at-e products ofarachidonate