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Regulation of differentiation of erythroid cells
BIOCHIMIE, 1972, 54, 607-811.
COLLOQUE C.N.R.S. BASES MOLECULAIRES DE LA PATItOLOGIE. 1 1 - 1 3 s e p t e m b r e 1971.
Regulation of differentiation of erythroid cells. P a u l A. MAnKS a n d R i c h a r d
A. RIFKIND.
Department o[ Medicine and o[ Human Genetics Development, Columbia Universil!t. College o[ Physicians and Surgeons, N e w York 10032. R~sum~. - - L e s p r o b l G m e s posGs p a r la d i f f G r e n e i a t i o n d e s c e l l u l e s 6 r y t h r o i d e s concernent : 1. L e s b a s e s e e l l u l a i r e s d u c h a n g e m e n t de s y n t h ~ s e de l ' h ~ m o g l o b i n e a u c o u r s d u d ~ v e l o p p e m e n t foetal et post-foetal ; 2. L e s , r e l a t i o n s e n t r e les l i g n ~ e s p r i m i t i v e s et d ~ f l n i t i v e s d e s c e l l u l e s ~ r y t h r o i d e s ; 3. L ' a c t i v i t ~ m i t o t i q u e d e s 6 r y t h r o b l a s t e s et 'la r e l a t i o n avee la s y n t h G s e de l ' h G m o gtobine ; 4. La s t a b i l i t 6 des R N A m e s s a g e r s en r e l a t i o n avec la s y n t h G s e d e s diffGrentes c l a s s e s de protGi'nes a u e o u r s de la d i f f G r e n e i a t i o n des e e l l u l e s d r y t h r o i d e s ; 5. L a n a t u r e de V a c t i o n de l ' G r y t h r o p o i d t i n e s u r la d'iffGrenciation celtu'laire et la s y n t h G s e d e l?hGmoglobine. Ce a) b) c) et la
d e r n i e r p r o b l G m e s e r a d i s c u t 6 p l u s en dGtail en a n a l y s a n t les donnGes s u r : La natu~re, de l a eel'lu.le qu.i rGpo~nd h l'aet~on de l'6rythr.opoiGtine, Le m 4 c a n i s m e p a r l e q u e l l ' G r y t h r o p o i G t i n e a u g m e n t e la f o r m a t i o n d ' h G m o g l o b i n e , L'effet de l ' G r y t h r o p o i G t i n e s u r le RNA, le DNA, la s y n t h G s e protGique, la rGplieation diffGreneiation eellulaire.
L'Grythro.poi~se c h e z le foetus de s o u r i s f o u r n i t u n m o d u l e p o u r l ' ~ t u d e de q u e l q u e s a s p e c t s i m p o r t a n t s de la r G g u l a t i o n de la d i f f G r e n e i a t i o n de la c e l l u l e et de la s y n t h G s e protGique. La m o d i f i c a t i o n de la n a t u r e d e s h G m o g l o b i n e s form4.es a u e o u r s d u dGvelopp e m e n t foetal e t p o s t - f o e t a l est associGe h la s u b s t i t u t i o n de lignGe d e s c e l l u l e s 6 r y t h r o i d e s d u foie. A u c o u r s de l a d i f f G r e n e i a t i o n d e s ce]lules 6 r y t h r o i d e s d u sac v i t e l l i n , on t r o u v e a u m o i n s d e u x c l a s s e s de p r o t G i n e s q u i s e d i . s t i n g u e n t p a r t e u r d G p e n d a n c e v i s - a - v i s d ' u n e f o r m a t i o n c o n t i n u e de RNA. L ' e n s e m b l e d e s p r o t G i n e s n u c l G a i r e s , p r o t G i n e s <>, e s t li~ h la s y n t h b s e d e s R N A m e s s a g e r s h durGc de v i e r e l a t i v e m . e n t c o u r t e a l o r s q u e la s y n t h G s e d e s protGines diffGrenciGes, les h G m o g l o b i n e s , e s t associGe h d e s R N A m e s s a g e r s r e l a t i v e m e n t s t a b l e s . L a f o r m a t i o n d ' h G m o g l o b i n e a l i e u d a n s ces c e l l u l e s q u i s y n t h G t i s e n t a c t i v e m e n t le DNA et se d i v i s e n t a c t i v e m e n t . E n v i r o n d e u x ou t r o i s d i v i s i o n s c e l l u l a i r e s se p r o & u i s e n t aprGs la f o r m a t i o n et la s t a b i l i s a t i o n d u R N A m e s s a g e r de la g l o b i n e . L ' G r y t h r o p o i G s e d u s a c v i t e l l i n , d u m o i n s h p a r t i r d u d i x i b m e j o u r de gestati(~n, ne r~pan.d ,pas h l ' ~ r y t h r o p o i G t i n e . P ~ r contr.e, l,ors d e l ' ~ r y t h r o p o i ~ s e da.ns le foie foetal, l ' ~ r y t h r o p o i G t i n e a g i t s ~ l e c t i v e m e n t s u r l e s c e l l u l e s ~ r y t h r o i d e s les p l u s i m m a t u r e s p o u r i n d u i r e l e u r d i f f ~ r e n c i a t i o n , la r ~ p l i c a t i o n c e l l u l a i r e et la f o r m a t i o n d ' h ~ m o g l o b i n e . L e s c e l l u l e s s e n s i b l e s h l ' ~ r y t h . r o p o i ~ t i n e d u foie s o n t a p p a r e m m e n t d i g fGrenci.~es h p a r t i r de la e e l l u l e s o u c h e p l u r i p o t e n t i e l l e d u foie e n t o r m a n t les 6ryt h r o b l a s f e s et e n s y n t h ~ t i . s a n t I ' h 5 m o g l o b i n e s o u s F a c t i o n de l ' h o r m o n e . L e s effets i n i t i a u x de l%rythropoiGti~ne s u r la syn.th~se macxomolficu'lair,e pro,Lent s u r l.a sti.mula.tion d e la s y ~ t h ~ s e de R N A q u i e s t s u i v i e de r g p l i c a t i o n c e l l u t a i r e et d ' u n e a u g m e n t a t i o n de f o r m a t i o n d ' h d m o g l o b i n e . L o r s de l'~ry.thropoi~se h ~ p a t i q u e , ii s e m b l e y a v o i r p a s s a g e de s y n t h G s e d'hdmog, R>bine d.~pendant de la fo,rma±ion de R N A /~ la s y n t h g s e d'hGmoglo,bi,ne h p a r t i r de RNA m e s s a g e r s r e l a t i v e m e n t s t a b l e s . A p r o p o s d u m G c a n i s m e d ' a c t i o n de l ' ~ r y t h r o p o i ~ t i n e , il est i n t ~ r e s s a n t de n o t e r q u e c e r t a i n e s s u b s t a n c e s q u i a g i s s e n t s S l e e t i v e m e n t s u r la e r o i s s a n e e c e l l u l a i r e , t e l l e q u e le f a c t e u r de e r o i s s a n c e n e r v e n x , la p h y t o h G m a g g l u t i n i n e , l ' i n s u l i n e , etc., s t i m u l e n t la s y n thGse d u RNA. On a su~gGr.fi qu.e eeei i m p l i q u . e u n e s u i t e con, s t a n t e d,e r~actions, b i o c h i m i q u e s en r S p o n s e ~t d e s s t i m u l i e x t ~ r i e u r s p e u t - ~ t r e p a r l ' i n t e r m 6 d i a i r e d ' u n e s u b s t a n c e m$diatrice commune. S u r la b a s e d e s r G s u l t a t s o b t e n u s , on p e u t s u g g ~ r e r l ' h y p o t h ~ s e s u i v a n t e : l ' G r y t h r o poiGtine a g i t s u r u n e c e l l u l e spGcifique, u n p r o ~ r y t h r o b l a s t e , q u i p r o v i e n t de la diffGrenelation d'une cellule souehe pluripotentielle. Cette cellule sensible h l'~ryth,ropoi~tine e s t p r o g r a m m G e p o u r se diffGreneier e n e e l l u l e s ~ r y t h r o i d e s c a p a b l e s de s y n t h ~ t i s e r l ' h G m o g l o b i n e . C e t t e c e l l u l e s e n s i b l e est e l l e - m ~ m e diffdrenciGe p a r le f a i t q u ' e l l e c o n t i e n t u n m $ c a n i ' s m e de r e o n n a l s s a n e e , p e u t - ~ t r e localis~ s u r la m e m b r a n e , q u i e x p l i q u e sa s e n s i b i l i t 6 sGlective h l ' h o r m o n e . L ' h o r m o n e e s t i n d i s p e n s a b l e p o u r i n d u i r e la t r a n s f o r m a t i o n
P a u l A . M a r k s a n d R i c h a r d A. R i f k i n d .
608
de la eellule sensible en 4rythroblastes eapabl.es de synth~tiser l'h~moglobine. L'effet primaire de l'hormone sur la synth~se de macromol6cules est la stimulation de la synth6sc de nouvell.es esp6ces mol6culaires de RNA dans la cellule sensible. La synth~se ainsi induite de RNA provoque h son tour la r6plication cellulaire, peut-~tre par l'intermddiaire de facteurs non encore identifi~s et dont la synth6se est d6termin6e par le RNA. Cette r6ponse peut repr~sen.ter un modble d'un processus plus g6n6ral concernant la croissance cellulaire et la diff6renciation sous contr61e hormonal. The r e g u l a t i o n of the d i f f e r e n t i a t i o n of eukaryotic cells is one of the m a j o r u n r e s o l v e d p r o b l e m s i n biology. An a p p r o a c h to the e l u c i d a t i o n of this p r o b l e m d e m a n d s a methodology for study of the p r e c u r s o r of differentiated cells. This i m m e d i a t e l y presents a p r o b l e m since we c a n n o t precisely define the u n d i f f e r e n t i a t e d cell. The differentiated cell is generally recognized as one w h i c h synthesizes specialized proteins. I n almost all a n i m a l cell p o p u l a t i o n s in vivo there is heterogeneity with regard to the cell type and w i t h respect to the stage of d i f f e r e n t i a t i o n of each specific cell type. Therefore, it becomes difficult to recognize, let alone analyze, the characteristics of p r e c u r s o r cells u n d e r in vivo conditions, since they generally constitute a small p r o p o r t i o n of the total cell population. I n studies on the e r y t h r o i d cell system and, more specifically, on fetal mouse liver erythropoiesis, there has been c o n s i d e r a b l e progress i n the isolation and c h a r a c t e r i z a t i o n of the p r e c u r s o r cell of erythroblasts. E r y t h r o i d cells p e r se afford a suitable model for the study of a s p e c t r u m of i m p o r t a n t questions related to the r e g u l a t i o n of cell differentiation. I n a d d i t i o n to a large b o d y of knowledge w i t h respect to the genetic, chemical, physiological a n d cytological aspects of e r y t h r o i d cells, one can employ the h o r m o n e e r y t h r o p o i e t i n i n o r d e r to study the process of i n d u c t i o n of erythropoiesis a n d h e m o g l o b i n synthesis. It m u s t be stated that, at present, there is no single ideal system for analysis of all or even most of the critical questions related to the r e g u l a t i o n of differentiation. Nevertheless, studies w i t h the fetal mouse e r y t h r o i d cell system have p r o v i d e d considerable insight into a n u m b e r of these questions. In this paper, we will review two of these quest i o n s : 1) The cellular basis for c h a n g i n g the p a t t e r n s of h e m o g l o b i n synthesis d u r i n g fetal a n d post-fetal development, and 2) The n a t u r e of the action of e r y t h r o p o i e t i n on cell differentiation a n d h e m o g l o b i n synthesis. A detailed r e v i e w of these areas as well as of other questions, has recently been p u b l i s h e d [1].
BIOCHIMIE, 1972, 54, n ° 5- 6.
C}~LLULAR BASIS FOR CHANGING PATTERNS OF HEMOGLOBIN SYNTHESIS. Changes in the types of hemoglobins synthesized d u r i n g fetal a n d post-fetal d e v e l o p m e n t have been described for the mouse, man, tadpole, chick, a n d a variety of other species [2-5]. Studies i n the mouse have p r o v i d e d p e r h a p s the most direct evidence on the r e l a t i o n s h i p b e t w e e n alterations in the cell line active in erythropoiesis and the type of h e m o g l o b i n formed [6]. I n the mouse, whose gestation p e r i o d is about 21 days, the first m o r p h o l o g i c a l l y i d e n t i f i a b l e sites of erythropoiesis are the blood islands of the yolk sac w h e r e erythropoiesis can be recognized by the eighth day of gestation. P r o l i f e r a t i o n of e r y t h r o i d cell p r e c u r s o r s is active in these sites u n t i l about the t e n i h gestational day [7]. These cells e n t e r the fetal c i r c u l a t i o n as nucleated erythroblasts by the n i n t h fetal day. F r o m the tenth day o n w a r d , f u r t h e r division a n d differentiation o'f these cells proceeds in the c i r c u l a t i o n . Mitosis m a y be observed through the t h i r t e e n t h fetal day. Ribosomes and polyribosomes, a b u n d a n t in early stages of these cells, progressively decrease i n c o n c e n t r a tion as the celIs accumulate hemoglobin. This process is a c c o m p a n i e d by progressive c o n d e n s a t i o n of the n u c l e a r c h r o m a t i n , shrinkage and disappear a n c e of the nucleolus and overall n u c l e a r p y c n o sis. The m a t u r e c i r c u l a t i n g yolk sac-derived erythrocyte is nucleated but devoid of cytoplasmic m i t o c h o n d r i a and ribosomes. Three h e m o g l o b i n s are formed in yolk sac e r y t h r o i d ceils of the C57BL/6J strain of mice a n d have been characterized as e m b r y o n i c h e m o g l o b i n E~, composed of x a n d y globin chains, e m b r y o n i c h e m o g l o b i n E m conlposed of (t a n d y globin chains, a n d e m b r y o nic h e m o g l o b i n iii, composed of n a n d z globin chains [6]. There is no detectable synthesis of globin and, hence, no adult h e m o g l o b i n (%~,_,) in these cells. D u r i n g the tenth day of gestation, a second site of erythropoiesis, the liver, becomes m o r p h o l o g i c a l l y detectable. E r y t h r o i d cell p r e c u r sors, a p p a r e n t l y derived from m e s e n c h y m a l cells adjacent to cords of hepatic epithelial cells of e n d o d e r m a l origin [8], give rise to hemocytoblasts and, i n turn, to p r o e r y t h r o b l a s t s w h i c h differentiate through a series of developmental stages, to n o n - n u c l e a t e d reticulocytes. T e r m i n a l differentia-
Regulation of differentiation of erythroid cells. tion of these e r y t h r o i d ceils is c h a r a c t e r i z e d by n u c l e a r e x p u l s i o n p r i o r to loss of the c y t o p l a s m i c r i b o s o m e s a n d m i t o c h o n d r i a . L i v e r e r y t h r o i d cells synthesize a single t y p e of h e m o g l o b i n w i t h a globin c o m p o s i t i o n i n d i s t i n g u i s h a b l e f r o m that of h e m o g l o b i n p r e s e n t in the adult of this species, d e s i g n a t e d ct a n d fl globin [6]. These d a t a establish that t h e r e are two d i s t i n c t p o p u l a t i o n s of e r y t h r o i d cells a p p e a r i n g at different times d u r i n g mouse fetal development. The change in p a t t e r n of h e m o g l o b i n s y n t h e s i s f r o m e m b r y o n i c to adult t y p e of h e m o g l o b i n is assoc i a t e d w i t h the substitution of l i v e r e r y t h r o p o i e s i s for y o l k sac e r y t h r o p o i e s i s . This cellular basis for c h a n g i n g p a t t e r n s of h e m o g l o b i n s y n t h e s i s a p p e a r s to be a m o r e general p h e n o m e n o n in animal development. E v i d e n c e i n d i c a t e s that an analogous shift from a p r i m i t i v e to definitive eryt h r o i d cell line is a s s o c i a t e d w i t h a change in t y p e s of h e m o g l o b i n s y n t h e s i z e d in the chick, man, a n d the t a d p o l e [2-5, 9, 10]. I n a d d i t i o n to this p a t t e r n of c e l l u l a r changes w h i c h d e t e r m i n e s changes in h e m o g l o b i n f o r m a t i o n , a f u r t h e r suggestion of p a t t e r n m a y be d i s c e r n e d in the struct u r a l alterations i n the h e m o g l o b i n s f o r m e d in p r i m i t i v e a n d d e f i n i t i v e cell lines. I n the mouse. man, a n d chicken, c o n v e r s i o n f r o m e m b r y o n i c to a d u l t h e m o g l o b i n involves the substitution of one globin c h a i n [2-5]. In the mouse and in m a n this substitution is in the beta-like chain. The alphac h a i n is constant in s t r u c t u r e in the e m b r y o n i c a n d adult h e m o g l o b i n s , p r e s u m a b l y r e f l e c t i n g c o n s t a n t genetic a c t i v i t y for the a l p h a c h a i n and a l t e r e d genetic a c t i v i t y for the b e t a - t y p e chain. In the c h i c k e n , likewise, one globin c h a i n a p p e a r s to be c o n s t a n t b e t w e e n the e m b r y o n i c an adult h e m o g l o b i n [5], although this is not the ease w i t h the t a d p o l e - f r o g system w h i c h have no c o m m o n p e p t i d e c h a i n s (9, 10]. PRECURSOR CELL OF F E T A L LIVER ERYTHROBLASTS. Central to o u r u n d e r s t a n d i n g of the r e g u l a t i o n of e r y t h r o i d cell d i f f e r e n t i a t i o n a n d h e m o g l o b i n s y n t h e s i s is i d e n t i f i c a t i o n of the cell in w h i c h the s y n t h e s i s of these p r o t e i n s is i n i t i a t e d . F r o m d a y 11 of gestation, the fetal l i v e r is a site of e r y t h r o p o i e s i s a n d the p o p u l a t i o n of l i v e r e r y t h r o i d cells becomes i n c r e a s i n g l y m o r e d i f f e r e n t i a t e d [8, 1115]. On d a y 11, a p p r o x i m a t e l y 80 p. cent of the e r y t h r o i d cells p r e s e n t in the l i v e r are of a v e r y i m m a t u r e stage, but b y d a y 14, the p r o p o r t i o n of these i m m a t u r e cells decreases s h a r p l y , to less t h a n 5 p. cent, a n d t h e r e is a c o n e m n i t a n t i n c r e a s e
BIOCH1MIE, 1972, 54,
n ° 5 - 6.
609
in the p r o p o r t i o n of h e m o g l o b i n i z e d , o r t h o c h r o m i c e r y t h r o b l a s t s [1]. D u r i n g this p e r i o d , t h e r e is a greater t h a n 10-fold i n c r e a s e in the total n u m b e r of e r y t h r o i d cells in the l i v e r [16]. E r y t h r o p o i e s i s in the fetal liver p r o c e e d s as a h e t e r o g e n e o u s p o p u l a t i o n w i t h r e g a r d to cell stage, in c o n t r a s t to y o l k sac e r y t h r o i d ceils w h i c h d i f f e r e n t i a t e in a r e l a t i v e l y h o m o g e n e o u s fashion. Thus, y o l k sac e r y t h r o i d cells a p p e a r to develop from p r e c u r s o r cells as a cohort, w h i l e l i v e r e r y t h r o p o i e s i s , at least for a s h o r t p e r i o d , involves a self-perpetuating p r e c u r s o r cell w h i c h y i e l d s d i f f e r e n t i a t i n g e r y t h r o b l a s t s over a p e r i o d of time. E r y t h r o poiesis in the liver, h o w e v e r , is a t r a n s i t i o n a l p r o cess ; b y birth, the l i v e r is no l o n g e r an active site of e r y t h r o p o i e s i s [17!. In 11-day fetal livers, there is a p o p u l a t i o n of cells, m o r p h o l o g i c a l l y classified as p r o e r y t h r o blasts, w h i c h s h o w a v e r y active i n c o r p o r a t i o n of u r i d i n e into RNA a n d l e u c i n e into p r o t e i n . This is i n d i c a t e d b y a r a t e of ~H-uridine u p t a k e a n d aH-leucine u p t a k e b y these cells w h i c h is 3 to 5-fold greater t h a n that in m o r p h o l o g i c a l l y comp a r a b l e cells on subsequent d a y s of gestation [1]. In a d d i t i o n to m o r p h o l o g i c a l c r i t e r i a , as w i l l be i n d i c a t e d below, e v i d e n c e that these cells are eryt h r o i d cell p r e c u r s o r s i n c l u d e s t h e i r selective resp o n s e to the p h y s i o l o g i c r e g u l a t o r of e r y t h r o poiesis, the h o r m o n e e r y f h r o p o i e t i n [18]. Its d i s a p p e a r a n c e a n d c o n s e q u e n t loss of the c a p a c i t y for s u s t a i n e d e r y t h r o p o i e s i s m a y a c c o u n t for the t r a n s i e n t n a t u r e of e r y t h r o p o i e s i s in the mousse fetal liver. T h e r e is c o n s i d e r a b l e e v i d e n c e that the p r e c u r sor cell r e s p o n s i v e to e r y t h r o p o i e t i n is d i s t i n c t f r o m the p l u r i p o t e n t i a l h e m a t o p o i e t i c stem cell. Thus, S t e p h e n s o n a n d A x e l r a d [19], e m p l o y i n g velocity sedimentation, separated erythropoietinr e s p o n s i v e cells from h e m a t o p o i e t i c spleen colonyf o r m i n g cells of mouse fetal liver. E a r l i e r biological studies p r o v i d e d e v i d e n c e that p l u r i p o t e n t i a l spleen c o l o n y - f o r m i n g cells are d i s t i n c t from (and p r o b a b l y i n c l u d e the p r o g e n i t o r of) the e r y t h r o p o i e t i n sensitive cell [20]. E F F E C T OF ERYTHROPOIETIN. The n a t u r e of the e r y t h r o p o i e t i n - r e s p o n s i v e cell, the i m m e d i a t e p r e c u r s o r of the h e m o g l o b i n - f o r m i n g cell, was e x a m i n e d f u r t h e r at the celhflar level. G o l d w a s s e r a n d c o w o r k e r s [21], s t u d i n g adult r a t m a r r o w , a n d Paul a n d c o w o r k e r s El1-13, 22, 23], s t u d y i n g fetal mouse l i v e r e r y t h r o i d cells, demonstrated that erythropoietin induces increased h e m o g l o b i n formation. In fetal liver e r y t h r o i d
P a u l A. M a r k s and R i c h a r d A. R i [ k i n d .
610
cells, the h o r m o n e - i n d u c e d i n c r e a s e in h e m o g l o b i n f o r m a t i o n does not result from a d i r e c t effect on the rate of h e m o g l o b i n s y n t h e s i s p e r cell [15]. This is i n d i c a t e d b y the o b s e r v a t i o n t h a t e r y t h r o p o i e t i n does not i n c r e a s e the u p t a k e of 3H-leucine p e r p o l y c h r o m a t o p h i l i e or p e r o r t h o c h r o m a t i c eryt h r o b l a s t in cultures in w h i c h the h o r m o n e causes a p p r o x i m a t e l y 2-fold i n c r e a s e in h o m o g l o b i n formation. The e r y t h r o p o i e t i n - s t i m u l a t e d i n c r e a s e in h e m o g l o b i n s y n t h e s i s is due to an i n c r e a s e in the n u m b e r of cells s y n t h e s i z i n g h e m o g l o b i n . I n c u b a tion of e r y t h r o i d ceils, d e r i v e d from fetal liver, w i t h e r y t h r o p o i e t i n for 24 h o u r s results in an a p p r o x i m a t e l y 2-fold i n c r e a s e in the n u m b e r of e r y t h r o b l a s t s c o m p a r e d w i t h cultures w i t h o u t e r y t h r o p o i e t i n . T h e h o r m o n e acts to m a i n t a i n the n u m b e r of i m m a t u r e p r o e r y t h r o b l a s t s a n d basop h i l i c e r y t h r o b l a s t s in the p o p u l a t i o n , a n d to i n c r e a s e the total n u m b e r of h e m o g l o b i n - f o r m i n g cells. E r y t h r o p o i e t i n is r e q u i r e d for r e n e w a l of the i m m a t u r e e r y t h r o i d cell p r e c u r s o r p o p u l a t i o n u n d e r these in vitro c o n d i t i o n s . Yolk sac e r y t h r o poiesis, in w h i c h i m m a t u r e p r e c u r s o r s do not persist, is not d e m o n s t r a b l y r e s p o n s i v e to e r y t h r o p o i e t i n [1, 11-13]. It s h o u l d be n o t e d that the resp o n s i v e n e s s of y o l k sac e r y t h r o i d cells w a s examined o n l y from d a y 10. It is c o n c e i v a b l e that at an e a r l i e r stage a p r e c u r s o r of these cells exists w h i c h is e r y t h r o p o i e t i n r e s p o n s i v e . This possibil i t y is r a i s e d b y the finding, s m n m a r i z e d l a t e r in this r e v i e w , that in the liver, o n l y the most immature e r y t h r o i d cell is r e s p o n s i v e to e r y t h r o p o i e t i n . Yolk sac e r y t h r o i d cells, u n l i k e l i v e r e r y t h r o i d cells, differentiate as a cohort, and a c o m p a r a b l e p r e c u r s o r cell m a y not be p r e s e n t after d a y 8 of gestation. These o b s e r v a t i o n s are c o n s i s t e n t w i t h studies in several l a b o r a t o r i e s [18] w h i c h suggest that the c o n t i n u o u s p r e s e n c e of e r y t h r o p o i e t i n is not n e c e s s a r y for e r y t h r o b l a s t m a t u r a t i o n but does stimulate erythropoiesis. I n fetal l i v e r e r y t h r o i d cells, the first d e t e c t a b l e effect of e r y t h r o p o i e t i n on m a c r o m o l e c u l a r synthesis is a s t i m u l a t i o n of the rate of RNA formation [15, 24-26]. R a d i o a u t o g r a p h i c studies demonst r a t e d that this effect of the h o r m o n e is selective, in t h a t it is r e s t r i c t e d in culture to a s t i m u l a t i o n of the rate of RNA s y n t h e s i s in the most i m m a t u r e e r y t h r o i d cells, the p r o e r y t h r o b l a s t s [15]. The h o r m o n e h a d no effect on RNA f o r m a t i o n in m o r e d i f f e r e n t i a t e d e r y t h r o i d cells or in n o n - e r y t h r o i d cells. These e r y t h r o p o i e t i n r e s p o n s i v e cells are includ e d in a class of p r e c u r s o r cells d e s i g n a t e d p r o e r y t h r o b l a s t s on the basis of c y t o l o g i c a l c r i t e r i a . The most i m m a t u r e e r y t h r o i d ceils in the fetal BIOCH1MIE,
1 9 7 2 , 54, n ° 5 - 6.
mouse l i v e r are n o m i n a l l y d e s i g n a t e d p r o e r y t h r o blasts. It is r e c o g n i z e d that c y t o l o g i c a l c r i t e r i a are i n a d e q u a t e to d i s t i n g u i s h the e r y t h r o p o i e t i n resp o n s i v e cells f r o m ceils of the first e r y t h r o b l a s t stage a l r e a d y c o m m i t t e d to h e m o g l o b i n s y n t h e s i s a n d differentiation. The cells in this p o p u l a t i o n are large and, b y light a n d e l e c t r o n m i c r o s c o p y , d i s p l a y a h i g h n u c l e a r to c y t o p l a s m i c r a t i o a n d an e x t e n d e d c h r o m a t i n p a t t e r n w i t h i n the large nucleus. The c y t o p l a s m c o n t a i n s a b u n d a n t p o l y ribosomes, m i t o c h o n d r i a , a n d s p a r s e elements of the e n d o p l a s m i c reticulum. In these cells, e r y t h r o p o i e t i n s t i m u l a t i o n of RNA s y n t h e s i s is not d e p e n d e n t on a n y h o r m o n e m e d i a ted effect on DNA s y n t h e s i s [27, 28]. I n h i b i t i o n of DNA s y n t h e s i s b y h y d r o x y u r e a or c y t o s i n e a r a b i noside, does not p r e v e n t the e r y t h r o p o i e t i n - s t i m u l a t e d RNA s y n t h e s i s in p r o e r y t h r o b l a s t s . Compar a b l e results w e r e r e p o r t e d for rat m a r r o w [28]. E r y t h r o p o i e t i n - s t i m u l a t e d RNA s y n t h e s i s in fetal liver p r o e r y t h r o b l a s t s in culture p r e c e d e s detectable i n c r e a s e in cell n u m b e r a n d in h e m o g l o b i n s y n t h e s i s [15, 27, 28]. The nature of the e r y t h r o p o i e t i n - s t i m u l a t e d I:tNA in fetal l i v e r e r y t h r o b l a s t s has not y e t been d e t e r m i n e d . I n r a t m a r r o w , Gross a n d G o l d w a s s e r [24-26] r e p o r t that e r y t h r o p o i e t i n causes stimulation of a v a r i e t y of I~NA s p e c i e s i n c l u d i n g 150S, 55-65S, 45S, 28S, 18:S, 9iS, 6S, and 4S. This is in c o n t r a s t to an e a r l i e r r e p o r t from the same labor a t o r y w h i c h c o n c l u d e d e r y t h r o i e t i n selectively s t i m u l a t e d 10-12S RNA a n d suggested that the h o r m o n e might be a c t i n g p r i m a r i l y b y i n c r e a s i n g s y n t h e s i s of m e s s e n g e r RNA for globin. HYPOTHESIS FOR A.CTION O F ERYTHROPOIETIN. In c o n s i d e r i n g the m e c h a n i s m s of action of e r y t h r o p o i e t i n , it is i n t e r e s t i n g to note that a numb e r of substances w h i c h affect, selectively, cell g r o w t h a n d differentiation, such as n e r v e g r o w t h factor [29], p h y t o h e m a g g l u t i n i n [3'0], i n s u l i n [31] a n d others [32], stimulate the s y n t h e s i s of RNA as the i n i t i a l affect on m a c r o m o l e c u l a r f o r m a t i o n . It has been suggested that this involves a c o n s t a n t set of b i o c h e m i c a l r e a c t i o n s r e s p o n d i n g c o o r d i n a t e l y to the e n v i r o n m e n t a l stimulus, p o s s i b l y via a comm o n m e d i a t o r substance [31]. On the basis of the p r e s e n t l y available data, the following h y p o t h e s i s is suggested (Fig. 1). E r y t h r o p o i e t i n (EJPO) acts on a specific cell, a p r o e r y t h r o b l a s t (E.R.C.), w h i c h is d i f f e r e n t i a t e d from a p r o g e n i t o r p l u r i p o t e n t i a l stem cell. This e r y t h r o -
R e g u l a t i o n o f d i f f e r e n t i a t i o n o f e r y t h r o i d cells. p o i e t i n - r e s p o n s i v e cell is p r o g r a m m e d to d i f f e r e n t i a t e to e r y t h r o i d c e l l s c a p a b l e of s y n t h e s i z i n g h e m o g l o b i n . T h e e r y t h r o p o i e t i n - r e s p o n s i v e cell is d i f f e r e n t i a t e d to h a v e a r e c e p t o r site, p o s s i b l y o n t h e m e m b r a n e f o r e r y t h r o p o i e t i n . T h e h o r m o n e is n e c e s s a r y to i n d u c e t h e t r a n s i t i o n f r o m t h e e r y thropoietin-responsive cell t o t h e e r y t h r o b l a s t s t a g e s c a p a b l e of h e m o g l o b i n s y n t h e s i s . T h e p r i -
MECHANISM OF ERYTHROPOIETIN ACTION EPO PLURIPOTENTIAL---> E . R . C , / "
CELL ~ (?)
--
PROTEIN(?)
'I' DNA
~'
MITOSIS
(71> ERYTHROBLAST
(Hg Synthesis) FIG. 1. Hypothesis as to the m e e h a n l s m of e r y t h r o p o i e t i n action. See text for detailed discussion of this model. A b b r e v i a t i o n s : E . R . C . - e r y t h r o p o i e t i n responsive eel1 w h i c h is identified b y cytological criteria as a p r o e r y t h r o b l a s t . E P O - - e r y t h r o p o i e t i n . Hg - - h e m o globin.
m a r y e f f e c t of t h e h o r m o n e o n m a c r o m o l e c u l a r s y n t h e s i s is t o s t i m u l a t e t h e f o r m a t i o n o f a v a r i e t y of s p e c i e s of R N A i n t h e e r y t h r o p o i e t i n - r e s p o n s i v e cell. T h e h o r m o n e - i n d u c e d R N A s y n t h e s i s i n t u r n l e a d s to cell r e p l i c a t i o n , p o s s i b l y m e d i a t e d b y y e t u n i d e n t i f i e d f a c t o r s w h o s e s y n t h e s i s is d e t e r m i n e d b y t h e RNA. T h i s r e s p o n s e m a y b e r e p r e s e n t a t i v e of a m o r e g e n e r a l p h e n o m e n o n , characterizing hormone-dependent cell differentiation. REFERENCES. 1. Marks, P. A. ~ Rifkind, R. A. (1972) Science, 175, 955. 2. Kovach, J. S., Marks, P. A., Russell, E. S. ,~ Epter, H. J. (1967) J. Mnl. Biol., 25, 131. 3. Ingrain, V. M. (1963) The Hemoglobin in Genetics and Evolution• C o l u m b i a U n i v e r s i t y Press, N.Y.
BIOCHIMIE, 1972, 54, n ° 5 - 6 .
611
4. Baglioni, C• & Sparks, C. E. (1963) Develop. Biol., 8, 272. 5. WHt, F. H. (19'6'7) Adv. Morphol., 6, 89. 6. F a n t o n i , A., Bank, A. & Marks, P. A. (1967) Science, 157, 1.327. 7. Attfield, M. (1951) J. Genet., 50, 250. 8. Rifkind, R. A., Chui, D. H. K. & Epler, H. (1969) J. Cell Biol., 40, 343. 9. Moss, B. & Ingram, V. M. (19r68) J. Mol. Biol., 32, 493. 10. Maniatis, G. M. & Ingrain, V. M. (1971) J. Cell Biol., 49, 373. 11. Cole, R. J. ~ Paul, J. (196.6) J. Embrgol. Exp. Morph., 15, 245. 12. Paul, J. ~ Hunter, J. A. (1969) J. Mol. Biol., 42, 31. 13• Paul, J., Conkie, D. a F r e s h n e y , R. I. (19~9~)Cell Tissue Kinet., 2, 283. 14. R[fkind, R. A., Chui, D., Djaldetti, M. • Marks, P. A. (196'9) Trans. Am. Assoc. of Phys., 82, 380. 15. Chui, D., Djaldetti, M., Marks, P. A. • Rifkind, R. A. (1971) J. Cell Biol., 51, 585. 16. Barker, J. E., Keena~, M. A. • R a p h a l s , L. (1969) J. • .Cell. Physiol., 74, 51. 17. Russell E. S. • Bernstein, S. E. (196,6) in Biology of the Laboratory Mouse, E. L. G~een, Ed. McOrawHill, N.Y., p. 351. 18. Krantz, S. B. ~ Jacobson, L. O. (1970) Erythropoietin and Regulation of Erythropoiesis. The U n i v e r s i t y of Chicago Press, Chicago, p. 118. 19. Stephenson, J. R. a Axelrad, A. A. (li971~ Blood, 37, 417. 20. McCulloch, E. A. (1970 ~) in Regulation of Hematopoiesis, A. S. Gordon Ed. Appl'eton-CenturyCrofts, N.Y., v. 1, p. 132. 21. Gold~casser, E. (1966) i n Current Topics in Developmental Biology, A. Monroy a n d A. A. Moscon a Ed. Academic Press, N.Y., p. 7!3. 22. Hunter, J. A. ~ Patti, J. (1969) J. Embryol. Exp. Morph., 21, 361. 23. Paul, J. a Hunter, J. A. (196~) Nature (London), 219, 1362. 24. Gross, M. a Gold~vasser, E. (19r69) Biochem., 8, 1795. 25. Krantz, S. B. ~ Gold~vasser, E. (19~65) Biochim. Binphys. Acta, 103, 325. 20. Gross, M. & G o l d ~ a s s e r , E. (19T7) J. Biol. Chem., 246, 2480. 27. Gross, M. ~ Goldwasser, E. (1970) J. Biol. Chem., 245, 1.632. 28. Preisler, H., Djaldetti, M., Marks, P. A. ~ Rifkind, R. A. J. Biol. Chem. (Ill Press). 29. Angeletti, P. U., Levi-Montalcini, R. • Calissano, P. (196&) Advan. Enzymol., 31, 51. 30. Kay, J. E• ~ H a n d m a k e r , S. D. (1970) Exp. Cell Bes., 63, 411. 31. H~ershko, A., Mamont, P., Shields, R. ~ Tomkins, G. M. Nature (In Press). 32. Tttrkington, R. W. (1~971) in Developmental Aspects of the Cell Cycle, I. L. Cameron, G. M. P a d i l l a a n d A. M. Z i m m e r m a n Eds Academic Press, N.Y., p. 315.
COLLOQUE C.N.R.S. BASES MOLECULAIRES DE LA PATItOLOGIE. 1 1 - 1 3 s e p t e m b r e 1971.
Regulation of differentiation of erythroid cells. P a u l A. MAnKS a n d R i c h a r d
A. RIFKIND.
Department o[ Medicine and o[ Human Genetics Development, Columbia Universil!t. College o[ Physicians and Surgeons, N e w York 10032. R~sum~. - - L e s p r o b l G m e s posGs p a r la d i f f G r e n e i a t i o n d e s c e l l u l e s 6 r y t h r o i d e s concernent : 1. L e s b a s e s e e l l u l a i r e s d u c h a n g e m e n t de s y n t h ~ s e de l ' h ~ m o g l o b i n e a u c o u r s d u d ~ v e l o p p e m e n t foetal et post-foetal ; 2. L e s , r e l a t i o n s e n t r e les l i g n ~ e s p r i m i t i v e s et d ~ f l n i t i v e s d e s c e l l u l e s ~ r y t h r o i d e s ; 3. L ' a c t i v i t ~ m i t o t i q u e d e s 6 r y t h r o b l a s t e s et 'la r e l a t i o n avee la s y n t h G s e de l ' h G m o gtobine ; 4. La s t a b i l i t 6 des R N A m e s s a g e r s en r e l a t i o n avec la s y n t h G s e d e s diffGrentes c l a s s e s de protGi'nes a u e o u r s de la d i f f G r e n e i a t i o n des e e l l u l e s d r y t h r o i d e s ; 5. L a n a t u r e de V a c t i o n de l ' G r y t h r o p o i d t i n e s u r la d'iffGrenciation celtu'laire et la s y n t h G s e d e l?hGmoglobine. Ce a) b) c) et la
d e r n i e r p r o b l G m e s e r a d i s c u t 6 p l u s en dGtail en a n a l y s a n t les donnGes s u r : La natu~re, de l a eel'lu.le qu.i rGpo~nd h l'aet~on de l'6rythr.opoiGtine, Le m 4 c a n i s m e p a r l e q u e l l ' G r y t h r o p o i G t i n e a u g m e n t e la f o r m a t i o n d ' h G m o g l o b i n e , L'effet de l ' G r y t h r o p o i G t i n e s u r le RNA, le DNA, la s y n t h G s e protGique, la rGplieation diffGreneiation eellulaire.
L'Grythro.poi~se c h e z le foetus de s o u r i s f o u r n i t u n m o d u l e p o u r l ' ~ t u d e de q u e l q u e s a s p e c t s i m p o r t a n t s de la r G g u l a t i o n de la d i f f G r e n e i a t i o n de la c e l l u l e et de la s y n t h G s e protGique. La m o d i f i c a t i o n de la n a t u r e d e s h G m o g l o b i n e s form4.es a u e o u r s d u dGvelopp e m e n t foetal e t p o s t - f o e t a l est associGe h la s u b s t i t u t i o n de lignGe d e s c e l l u l e s 6 r y t h r o i d e s d u foie. A u c o u r s de l a d i f f G r e n e i a t i o n d e s ce]lules 6 r y t h r o i d e s d u sac v i t e l l i n , on t r o u v e a u m o i n s d e u x c l a s s e s de p r o t G i n e s q u i s e d i . s t i n g u e n t p a r t e u r d G p e n d a n c e v i s - a - v i s d ' u n e f o r m a t i o n c o n t i n u e de RNA. L ' e n s e m b l e d e s p r o t G i n e s n u c l G a i r e s , p r o t G i n e s <
P a u l A . M a r k s a n d R i c h a r d A. R i f k i n d .
608
de la eellule sensible en 4rythroblastes eapabl.es de synth~tiser l'h~moglobine. L'effet primaire de l'hormone sur la synth~se de macromol6cules est la stimulation de la synth6sc de nouvell.es esp6ces mol6culaires de RNA dans la cellule sensible. La synth~se ainsi induite de RNA provoque h son tour la r6plication cellulaire, peut-~tre par l'intermddiaire de facteurs non encore identifi~s et dont la synth6se est d6termin6e par le RNA. Cette r6ponse peut repr~sen.ter un modble d'un processus plus g6n6ral concernant la croissance cellulaire et la diff6renciation sous contr61e hormonal. The r e g u l a t i o n of the d i f f e r e n t i a t i o n of eukaryotic cells is one of the m a j o r u n r e s o l v e d p r o b l e m s i n biology. An a p p r o a c h to the e l u c i d a t i o n of this p r o b l e m d e m a n d s a methodology for study of the p r e c u r s o r of differentiated cells. This i m m e d i a t e l y presents a p r o b l e m since we c a n n o t precisely define the u n d i f f e r e n t i a t e d cell. The differentiated cell is generally recognized as one w h i c h synthesizes specialized proteins. I n almost all a n i m a l cell p o p u l a t i o n s in vivo there is heterogeneity with regard to the cell type and w i t h respect to the stage of d i f f e r e n t i a t i o n of each specific cell type. Therefore, it becomes difficult to recognize, let alone analyze, the characteristics of p r e c u r s o r cells u n d e r in vivo conditions, since they generally constitute a small p r o p o r t i o n of the total cell population. I n studies on the e r y t h r o i d cell system and, more specifically, on fetal mouse liver erythropoiesis, there has been c o n s i d e r a b l e progress i n the isolation and c h a r a c t e r i z a t i o n of the p r e c u r s o r cell of erythroblasts. E r y t h r o i d cells p e r se afford a suitable model for the study of a s p e c t r u m of i m p o r t a n t questions related to the r e g u l a t i o n of cell differentiation. I n a d d i t i o n to a large b o d y of knowledge w i t h respect to the genetic, chemical, physiological a n d cytological aspects of e r y t h r o i d cells, one can employ the h o r m o n e e r y t h r o p o i e t i n i n o r d e r to study the process of i n d u c t i o n of erythropoiesis a n d h e m o g l o b i n synthesis. It m u s t be stated that, at present, there is no single ideal system for analysis of all or even most of the critical questions related to the r e g u l a t i o n of differentiation. Nevertheless, studies w i t h the fetal mouse e r y t h r o i d cell system have p r o v i d e d considerable insight into a n u m b e r of these questions. In this paper, we will review two of these quest i o n s : 1) The cellular basis for c h a n g i n g the p a t t e r n s of h e m o g l o b i n synthesis d u r i n g fetal a n d post-fetal development, and 2) The n a t u r e of the action of e r y t h r o p o i e t i n on cell differentiation a n d h e m o g l o b i n synthesis. A detailed r e v i e w of these areas as well as of other questions, has recently been p u b l i s h e d [1].
BIOCHIMIE, 1972, 54, n ° 5- 6.
C}~LLULAR BASIS FOR CHANGING PATTERNS OF HEMOGLOBIN SYNTHESIS. Changes in the types of hemoglobins synthesized d u r i n g fetal a n d post-fetal d e v e l o p m e n t have been described for the mouse, man, tadpole, chick, a n d a variety of other species [2-5]. Studies i n the mouse have p r o v i d e d p e r h a p s the most direct evidence on the r e l a t i o n s h i p b e t w e e n alterations in the cell line active in erythropoiesis and the type of h e m o g l o b i n formed [6]. I n the mouse, whose gestation p e r i o d is about 21 days, the first m o r p h o l o g i c a l l y i d e n t i f i a b l e sites of erythropoiesis are the blood islands of the yolk sac w h e r e erythropoiesis can be recognized by the eighth day of gestation. P r o l i f e r a t i o n of e r y t h r o i d cell p r e c u r s o r s is active in these sites u n t i l about the t e n i h gestational day [7]. These cells e n t e r the fetal c i r c u l a t i o n as nucleated erythroblasts by the n i n t h fetal day. F r o m the tenth day o n w a r d , f u r t h e r division a n d differentiation o'f these cells proceeds in the c i r c u l a t i o n . Mitosis m a y be observed through the t h i r t e e n t h fetal day. Ribosomes and polyribosomes, a b u n d a n t in early stages of these cells, progressively decrease i n c o n c e n t r a tion as the celIs accumulate hemoglobin. This process is a c c o m p a n i e d by progressive c o n d e n s a t i o n of the n u c l e a r c h r o m a t i n , shrinkage and disappear a n c e of the nucleolus and overall n u c l e a r p y c n o sis. The m a t u r e c i r c u l a t i n g yolk sac-derived erythrocyte is nucleated but devoid of cytoplasmic m i t o c h o n d r i a and ribosomes. Three h e m o g l o b i n s are formed in yolk sac e r y t h r o i d ceils of the C57BL/6J strain of mice a n d have been characterized as e m b r y o n i c h e m o g l o b i n E~, composed of x a n d y globin chains, e m b r y o n i c h e m o g l o b i n E m conlposed of (t a n d y globin chains, a n d e m b r y o nic h e m o g l o b i n iii, composed of n a n d z globin chains [6]. There is no detectable synthesis of globin and, hence, no adult h e m o g l o b i n (%~,_,) in these cells. D u r i n g the tenth day of gestation, a second site of erythropoiesis, the liver, becomes m o r p h o l o g i c a l l y detectable. E r y t h r o i d cell p r e c u r sors, a p p a r e n t l y derived from m e s e n c h y m a l cells adjacent to cords of hepatic epithelial cells of e n d o d e r m a l origin [8], give rise to hemocytoblasts and, i n turn, to p r o e r y t h r o b l a s t s w h i c h differentiate through a series of developmental stages, to n o n - n u c l e a t e d reticulocytes. T e r m i n a l differentia-
Regulation of differentiation of erythroid cells. tion of these e r y t h r o i d ceils is c h a r a c t e r i z e d by n u c l e a r e x p u l s i o n p r i o r to loss of the c y t o p l a s m i c r i b o s o m e s a n d m i t o c h o n d r i a . L i v e r e r y t h r o i d cells synthesize a single t y p e of h e m o g l o b i n w i t h a globin c o m p o s i t i o n i n d i s t i n g u i s h a b l e f r o m that of h e m o g l o b i n p r e s e n t in the adult of this species, d e s i g n a t e d ct a n d fl globin [6]. These d a t a establish that t h e r e are two d i s t i n c t p o p u l a t i o n s of e r y t h r o i d cells a p p e a r i n g at different times d u r i n g mouse fetal development. The change in p a t t e r n of h e m o g l o b i n s y n t h e s i s f r o m e m b r y o n i c to adult t y p e of h e m o g l o b i n is assoc i a t e d w i t h the substitution of l i v e r e r y t h r o p o i e s i s for y o l k sac e r y t h r o p o i e s i s . This cellular basis for c h a n g i n g p a t t e r n s of h e m o g l o b i n s y n t h e s i s a p p e a r s to be a m o r e general p h e n o m e n o n in animal development. E v i d e n c e i n d i c a t e s that an analogous shift from a p r i m i t i v e to definitive eryt h r o i d cell line is a s s o c i a t e d w i t h a change in t y p e s of h e m o g l o b i n s y n t h e s i z e d in the chick, man, a n d the t a d p o l e [2-5, 9, 10]. I n a d d i t i o n to this p a t t e r n of c e l l u l a r changes w h i c h d e t e r m i n e s changes in h e m o g l o b i n f o r m a t i o n , a f u r t h e r suggestion of p a t t e r n m a y be d i s c e r n e d in the struct u r a l alterations i n the h e m o g l o b i n s f o r m e d in p r i m i t i v e a n d d e f i n i t i v e cell lines. I n the mouse. man, a n d chicken, c o n v e r s i o n f r o m e m b r y o n i c to a d u l t h e m o g l o b i n involves the substitution of one globin c h a i n [2-5]. In the mouse and in m a n this substitution is in the beta-like chain. The alphac h a i n is constant in s t r u c t u r e in the e m b r y o n i c a n d adult h e m o g l o b i n s , p r e s u m a b l y r e f l e c t i n g c o n s t a n t genetic a c t i v i t y for the a l p h a c h a i n and a l t e r e d genetic a c t i v i t y for the b e t a - t y p e chain. In the c h i c k e n , likewise, one globin c h a i n a p p e a r s to be c o n s t a n t b e t w e e n the e m b r y o n i c an adult h e m o g l o b i n [5], although this is not the ease w i t h the t a d p o l e - f r o g system w h i c h have no c o m m o n p e p t i d e c h a i n s (9, 10]. PRECURSOR CELL OF F E T A L LIVER ERYTHROBLASTS. Central to o u r u n d e r s t a n d i n g of the r e g u l a t i o n of e r y t h r o i d cell d i f f e r e n t i a t i o n a n d h e m o g l o b i n s y n t h e s i s is i d e n t i f i c a t i o n of the cell in w h i c h the s y n t h e s i s of these p r o t e i n s is i n i t i a t e d . F r o m d a y 11 of gestation, the fetal l i v e r is a site of e r y t h r o p o i e s i s a n d the p o p u l a t i o n of l i v e r e r y t h r o i d cells becomes i n c r e a s i n g l y m o r e d i f f e r e n t i a t e d [8, 1115]. On d a y 11, a p p r o x i m a t e l y 80 p. cent of the e r y t h r o i d cells p r e s e n t in the l i v e r are of a v e r y i m m a t u r e stage, but b y d a y 14, the p r o p o r t i o n of these i m m a t u r e cells decreases s h a r p l y , to less t h a n 5 p. cent, a n d t h e r e is a c o n e m n i t a n t i n c r e a s e
BIOCH1MIE, 1972, 54,
n ° 5 - 6.
609
in the p r o p o r t i o n of h e m o g l o b i n i z e d , o r t h o c h r o m i c e r y t h r o b l a s t s [1]. D u r i n g this p e r i o d , t h e r e is a greater t h a n 10-fold i n c r e a s e in the total n u m b e r of e r y t h r o i d cells in the l i v e r [16]. E r y t h r o p o i e s i s in the fetal liver p r o c e e d s as a h e t e r o g e n e o u s p o p u l a t i o n w i t h r e g a r d to cell stage, in c o n t r a s t to y o l k sac e r y t h r o i d ceils w h i c h d i f f e r e n t i a t e in a r e l a t i v e l y h o m o g e n e o u s fashion. Thus, y o l k sac e r y t h r o i d cells a p p e a r to develop from p r e c u r s o r cells as a cohort, w h i l e l i v e r e r y t h r o p o i e s i s , at least for a s h o r t p e r i o d , involves a self-perpetuating p r e c u r s o r cell w h i c h y i e l d s d i f f e r e n t i a t i n g e r y t h r o b l a s t s over a p e r i o d of time. E r y t h r o poiesis in the liver, h o w e v e r , is a t r a n s i t i o n a l p r o cess ; b y birth, the l i v e r is no l o n g e r an active site of e r y t h r o p o i e s i s [17!. In 11-day fetal livers, there is a p o p u l a t i o n of cells, m o r p h o l o g i c a l l y classified as p r o e r y t h r o blasts, w h i c h s h o w a v e r y active i n c o r p o r a t i o n of u r i d i n e into RNA a n d l e u c i n e into p r o t e i n . This is i n d i c a t e d b y a r a t e of ~H-uridine u p t a k e a n d aH-leucine u p t a k e b y these cells w h i c h is 3 to 5-fold greater t h a n that in m o r p h o l o g i c a l l y comp a r a b l e cells on subsequent d a y s of gestation [1]. In a d d i t i o n to m o r p h o l o g i c a l c r i t e r i a , as w i l l be i n d i c a t e d below, e v i d e n c e that these cells are eryt h r o i d cell p r e c u r s o r s i n c l u d e s t h e i r selective resp o n s e to the p h y s i o l o g i c r e g u l a t o r of e r y t h r o poiesis, the h o r m o n e e r y f h r o p o i e t i n [18]. Its d i s a p p e a r a n c e a n d c o n s e q u e n t loss of the c a p a c i t y for s u s t a i n e d e r y t h r o p o i e s i s m a y a c c o u n t for the t r a n s i e n t n a t u r e of e r y t h r o p o i e s i s in the mousse fetal liver. T h e r e is c o n s i d e r a b l e e v i d e n c e that the p r e c u r sor cell r e s p o n s i v e to e r y t h r o p o i e t i n is d i s t i n c t f r o m the p l u r i p o t e n t i a l h e m a t o p o i e t i c stem cell. Thus, S t e p h e n s o n a n d A x e l r a d [19], e m p l o y i n g velocity sedimentation, separated erythropoietinr e s p o n s i v e cells from h e m a t o p o i e t i c spleen colonyf o r m i n g cells of mouse fetal liver. E a r l i e r biological studies p r o v i d e d e v i d e n c e that p l u r i p o t e n t i a l spleen c o l o n y - f o r m i n g cells are d i s t i n c t from (and p r o b a b l y i n c l u d e the p r o g e n i t o r of) the e r y t h r o p o i e t i n sensitive cell [20]. E F F E C T OF ERYTHROPOIETIN. The n a t u r e of the e r y t h r o p o i e t i n - r e s p o n s i v e cell, the i m m e d i a t e p r e c u r s o r of the h e m o g l o b i n - f o r m i n g cell, was e x a m i n e d f u r t h e r at the celhflar level. G o l d w a s s e r a n d c o w o r k e r s [21], s t u d i n g adult r a t m a r r o w , a n d Paul a n d c o w o r k e r s El1-13, 22, 23], s t u d y i n g fetal mouse l i v e r e r y t h r o i d cells, demonstrated that erythropoietin induces increased h e m o g l o b i n formation. In fetal liver e r y t h r o i d
P a u l A. M a r k s and R i c h a r d A. R i [ k i n d .
610
cells, the h o r m o n e - i n d u c e d i n c r e a s e in h e m o g l o b i n f o r m a t i o n does not result from a d i r e c t effect on the rate of h e m o g l o b i n s y n t h e s i s p e r cell [15]. This is i n d i c a t e d b y the o b s e r v a t i o n t h a t e r y t h r o p o i e t i n does not i n c r e a s e the u p t a k e of 3H-leucine p e r p o l y c h r o m a t o p h i l i e or p e r o r t h o c h r o m a t i c eryt h r o b l a s t in cultures in w h i c h the h o r m o n e causes a p p r o x i m a t e l y 2-fold i n c r e a s e in h o m o g l o b i n formation. The e r y t h r o p o i e t i n - s t i m u l a t e d i n c r e a s e in h e m o g l o b i n s y n t h e s i s is due to an i n c r e a s e in the n u m b e r of cells s y n t h e s i z i n g h e m o g l o b i n . I n c u b a tion of e r y t h r o i d ceils, d e r i v e d from fetal liver, w i t h e r y t h r o p o i e t i n for 24 h o u r s results in an a p p r o x i m a t e l y 2-fold i n c r e a s e in the n u m b e r of e r y t h r o b l a s t s c o m p a r e d w i t h cultures w i t h o u t e r y t h r o p o i e t i n . T h e h o r m o n e acts to m a i n t a i n the n u m b e r of i m m a t u r e p r o e r y t h r o b l a s t s a n d basop h i l i c e r y t h r o b l a s t s in the p o p u l a t i o n , a n d to i n c r e a s e the total n u m b e r of h e m o g l o b i n - f o r m i n g cells. E r y t h r o p o i e t i n is r e q u i r e d for r e n e w a l of the i m m a t u r e e r y t h r o i d cell p r e c u r s o r p o p u l a t i o n u n d e r these in vitro c o n d i t i o n s . Yolk sac e r y t h r o poiesis, in w h i c h i m m a t u r e p r e c u r s o r s do not persist, is not d e m o n s t r a b l y r e s p o n s i v e to e r y t h r o p o i e t i n [1, 11-13]. It s h o u l d be n o t e d that the resp o n s i v e n e s s of y o l k sac e r y t h r o i d cells w a s examined o n l y from d a y 10. It is c o n c e i v a b l e that at an e a r l i e r stage a p r e c u r s o r of these cells exists w h i c h is e r y t h r o p o i e t i n r e s p o n s i v e . This possibil i t y is r a i s e d b y the finding, s m n m a r i z e d l a t e r in this r e v i e w , that in the liver, o n l y the most immature e r y t h r o i d cell is r e s p o n s i v e to e r y t h r o p o i e t i n . Yolk sac e r y t h r o i d cells, u n l i k e l i v e r e r y t h r o i d cells, differentiate as a cohort, and a c o m p a r a b l e p r e c u r s o r cell m a y not be p r e s e n t after d a y 8 of gestation. These o b s e r v a t i o n s are c o n s i s t e n t w i t h studies in several l a b o r a t o r i e s [18] w h i c h suggest that the c o n t i n u o u s p r e s e n c e of e r y t h r o p o i e t i n is not n e c e s s a r y for e r y t h r o b l a s t m a t u r a t i o n but does stimulate erythropoiesis. I n fetal l i v e r e r y t h r o i d cells, the first d e t e c t a b l e effect of e r y t h r o p o i e t i n on m a c r o m o l e c u l a r synthesis is a s t i m u l a t i o n of the rate of RNA formation [15, 24-26]. R a d i o a u t o g r a p h i c studies demonst r a t e d that this effect of the h o r m o n e is selective, in t h a t it is r e s t r i c t e d in culture to a s t i m u l a t i o n of the rate of RNA s y n t h e s i s in the most i m m a t u r e e r y t h r o i d cells, the p r o e r y t h r o b l a s t s [15]. The h o r m o n e h a d no effect on RNA f o r m a t i o n in m o r e d i f f e r e n t i a t e d e r y t h r o i d cells or in n o n - e r y t h r o i d cells. These e r y t h r o p o i e t i n r e s p o n s i v e cells are includ e d in a class of p r e c u r s o r cells d e s i g n a t e d p r o e r y t h r o b l a s t s on the basis of c y t o l o g i c a l c r i t e r i a . The most i m m a t u r e e r y t h r o i d ceils in the fetal BIOCH1MIE,
1 9 7 2 , 54, n ° 5 - 6.
mouse l i v e r are n o m i n a l l y d e s i g n a t e d p r o e r y t h r o blasts. It is r e c o g n i z e d that c y t o l o g i c a l c r i t e r i a are i n a d e q u a t e to d i s t i n g u i s h the e r y t h r o p o i e t i n resp o n s i v e cells f r o m ceils of the first e r y t h r o b l a s t stage a l r e a d y c o m m i t t e d to h e m o g l o b i n s y n t h e s i s a n d differentiation. The cells in this p o p u l a t i o n are large and, b y light a n d e l e c t r o n m i c r o s c o p y , d i s p l a y a h i g h n u c l e a r to c y t o p l a s m i c r a t i o a n d an e x t e n d e d c h r o m a t i n p a t t e r n w i t h i n the large nucleus. The c y t o p l a s m c o n t a i n s a b u n d a n t p o l y ribosomes, m i t o c h o n d r i a , a n d s p a r s e elements of the e n d o p l a s m i c reticulum. In these cells, e r y t h r o p o i e t i n s t i m u l a t i o n of RNA s y n t h e s i s is not d e p e n d e n t on a n y h o r m o n e m e d i a ted effect on DNA s y n t h e s i s [27, 28]. I n h i b i t i o n of DNA s y n t h e s i s b y h y d r o x y u r e a or c y t o s i n e a r a b i noside, does not p r e v e n t the e r y t h r o p o i e t i n - s t i m u l a t e d RNA s y n t h e s i s in p r o e r y t h r o b l a s t s . Compar a b l e results w e r e r e p o r t e d for rat m a r r o w [28]. E r y t h r o p o i e t i n - s t i m u l a t e d RNA s y n t h e s i s in fetal liver p r o e r y t h r o b l a s t s in culture p r e c e d e s detectable i n c r e a s e in cell n u m b e r a n d in h e m o g l o b i n s y n t h e s i s [15, 27, 28]. The nature of the e r y t h r o p o i e t i n - s t i m u l a t e d I:tNA in fetal l i v e r e r y t h r o b l a s t s has not y e t been d e t e r m i n e d . I n r a t m a r r o w , Gross a n d G o l d w a s s e r [24-26] r e p o r t that e r y t h r o p o i e t i n causes stimulation of a v a r i e t y of I~NA s p e c i e s i n c l u d i n g 150S, 55-65S, 45S, 28S, 18:S, 9iS, 6S, and 4S. This is in c o n t r a s t to an e a r l i e r r e p o r t from the same labor a t o r y w h i c h c o n c l u d e d e r y t h r o i e t i n selectively s t i m u l a t e d 10-12S RNA a n d suggested that the h o r m o n e might be a c t i n g p r i m a r i l y b y i n c r e a s i n g s y n t h e s i s of m e s s e n g e r RNA for globin. HYPOTHESIS FOR A.CTION O F ERYTHROPOIETIN. In c o n s i d e r i n g the m e c h a n i s m s of action of e r y t h r o p o i e t i n , it is i n t e r e s t i n g to note that a numb e r of substances w h i c h affect, selectively, cell g r o w t h a n d differentiation, such as n e r v e g r o w t h factor [29], p h y t o h e m a g g l u t i n i n [3'0], i n s u l i n [31] a n d others [32], stimulate the s y n t h e s i s of RNA as the i n i t i a l affect on m a c r o m o l e c u l a r f o r m a t i o n . It has been suggested that this involves a c o n s t a n t set of b i o c h e m i c a l r e a c t i o n s r e s p o n d i n g c o o r d i n a t e l y to the e n v i r o n m e n t a l stimulus, p o s s i b l y via a comm o n m e d i a t o r substance [31]. On the basis of the p r e s e n t l y available data, the following h y p o t h e s i s is suggested (Fig. 1). E r y t h r o p o i e t i n (EJPO) acts on a specific cell, a p r o e r y t h r o b l a s t (E.R.C.), w h i c h is d i f f e r e n t i a t e d from a p r o g e n i t o r p l u r i p o t e n t i a l stem cell. This e r y t h r o -
R e g u l a t i o n o f d i f f e r e n t i a t i o n o f e r y t h r o i d cells. p o i e t i n - r e s p o n s i v e cell is p r o g r a m m e d to d i f f e r e n t i a t e to e r y t h r o i d c e l l s c a p a b l e of s y n t h e s i z i n g h e m o g l o b i n . T h e e r y t h r o p o i e t i n - r e s p o n s i v e cell is d i f f e r e n t i a t e d to h a v e a r e c e p t o r site, p o s s i b l y o n t h e m e m b r a n e f o r e r y t h r o p o i e t i n . T h e h o r m o n e is n e c e s s a r y to i n d u c e t h e t r a n s i t i o n f r o m t h e e r y thropoietin-responsive cell t o t h e e r y t h r o b l a s t s t a g e s c a p a b l e of h e m o g l o b i n s y n t h e s i s . T h e p r i -
MECHANISM OF ERYTHROPOIETIN ACTION EPO PLURIPOTENTIAL---> E . R . C , / "
CELL ~ (?)
--
PROTEIN(?)
'I' DNA
~'
MITOSIS
(71> ERYTHROBLAST
(Hg Synthesis) FIG. 1. Hypothesis as to the m e e h a n l s m of e r y t h r o p o i e t i n action. See text for detailed discussion of this model. A b b r e v i a t i o n s : E . R . C . - e r y t h r o p o i e t i n responsive eel1 w h i c h is identified b y cytological criteria as a p r o e r y t h r o b l a s t . E P O - - e r y t h r o p o i e t i n . Hg - - h e m o globin.
m a r y e f f e c t of t h e h o r m o n e o n m a c r o m o l e c u l a r s y n t h e s i s is t o s t i m u l a t e t h e f o r m a t i o n o f a v a r i e t y of s p e c i e s of R N A i n t h e e r y t h r o p o i e t i n - r e s p o n s i v e cell. T h e h o r m o n e - i n d u c e d R N A s y n t h e s i s i n t u r n l e a d s to cell r e p l i c a t i o n , p o s s i b l y m e d i a t e d b y y e t u n i d e n t i f i e d f a c t o r s w h o s e s y n t h e s i s is d e t e r m i n e d b y t h e RNA. T h i s r e s p o n s e m a y b e r e p r e s e n t a t i v e of a m o r e g e n e r a l p h e n o m e n o n , characterizing hormone-dependent cell differentiation. REFERENCES. 1. Marks, P. A. ~ Rifkind, R. A. (1972) Science, 175, 955. 2. Kovach, J. S., Marks, P. A., Russell, E. S. ,~ Epter, H. J. (1967) J. Mnl. Biol., 25, 131. 3. Ingrain, V. M. (1963) The Hemoglobin in Genetics and Evolution• C o l u m b i a U n i v e r s i t y Press, N.Y.
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611
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