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Hormonal control of cell-to-cell communication and of connexin gene expression in the thyroid
Biol. Cell, V o l 76, 1992
219
I N F L U E N C E OF T H E O N C O P R O T E I N v - e r b A ON Q U A I L MYOBLAST P R O L I F E R A T I O N A N D D I F F E R E N T I A T I O N .
D I F F E R E N T I A T I O N OF THE R A B B I T EMBRYO C O R N E A L E P I T H E L I U M : E X P R E S S I O N OF T H E A C I D I C KERATIN K12.
C A S S A R - M A L E K l,~abelle, MARCHAL S o p h i e , WRUTN1AK C h a n t a l , SAMARUT d a e q u e s * , CABELLO G 6 r a r d . U n i t 6 d e D l f f 6 r e n c l a t l o n C e l l u l a i r e et C r o l s s a n c e , 1NRA. P l a c e Viala, 3 4 0 6 0 MONTPELLIER C e d e x 1. * Lab. Biologic M o l 6 c u l a i r e et C e l l u l a l r e , C N R S U M R 4 9 , INRA E N S Lyon, 4 6 all6e tl'ltalle, 6 9 3 6 4 Lyon C~dex 0 7
CHALOIN-DUFAU Catherin~ 1, SUN Tung-Tien2 and DHOUAILLY Danielle1. 1- Laboratoire de Biologie de la Diffdrenciation Epithdliale. Universitd Joseph Fourier. CERMO. BP 53X. 38041 GRENOBLE. 2- Epithelial Biology Unit. Department of Dermatology. New York Universi~/ School of Medicine. NEW YORK.
N u m e r o u s d a t a e v i d e n c e t h a t t h e e x p r e s s i o n of t h e ve r b A o n e o g e n e I n h l b l t s t h e d i f f e r e n t i a t i o n of e r y t b r o l d cell p r e c u r s o r s (1) a n d t h e e x p r e s s i o n of s p e c i f i c e r y t h r o c y t l c g e n e s (2). It Is g e n e r a l l y a s s u m e d t h a t t h e v - e r b A p r o t e i n Is a b l e to a n t a g o n i z e t h e effects of t h e T 3 / n u c l e a r r e c e p t o r complexes on gene transcription. As we h a v e s h o w n t h a t T3 i n f l u e n c e s q u a i l m y o b l a s t p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n (3), we h a v e s t u d i e d t h e I n f l u e n c e of v - e r b A In o u r c u l t u r e model. We h a v e I n f e c t e d myoblasts with a recombinant retrovirus carrying the v-erbA o n c o g e n e . In t h e a b s e n c e of T3 ( a n d p r o b a b l y of r e t l n o i c acid), v - e r b A s i g n i f i c a n t l y I n c r e a s e s t h e p r o l i f e r a t i o n r a t e of myoblasts. Moreover. It e n h a n c e s accumulation of acetylcholine r e c e p t o r s a n d of m y o s i n h e a v y c h a i n , measured as differentiation markers. These results bring the first evidence that v-erbA could affect cell proliferation and differentiation I n d e p e n d e n t l y of T3 a n d r e t l n o l c acid. T h e y r a i s e also t h e following q u e s t i o n s : Is v - e r b A a b l e to s t i m u l a t e m y o b l a s t d i f f e r e n t i a t i o n ? A l t e r n a t i v e l y , d o e s It f a v o u r t h e e m e r g e n c e of m y o b l a s t s w i t h h i g h d i f f e r e n t i a t i o n p o t e n t i a l i t i e s , from a heterogencous myoblast population?
The K3/K12 keratins pair serves as biochemical marker for an advanced slage of "corneal-type" epithelial differenliation (Sun et al., 1984). Using an AK12 monoclonal antibody (Chaloin-Dufau et aL, 1990) highly specific for the acidic keratin K12, we have isolated a cDNA of this keralin from a ~.gll 1 rabbit corneal epithelium library. Sequential expression of the K12 keratin and of its mRNA was studied during embryonic d e v e l o p m e n t using bolh immunofluorescent slaining and in situ hybridization. The appearance of the protein and lhe activation of the corresponding gene are strictly synchronized: they appear first in Ihe suprabasal layer of central corneal epithelium at 17 days of gestation, and reach the basal epithelial layer six days later (23 days of gestation). Therefore, the regulation of the acidic keratin K12 gene appears to be primarily at the transcriptional level. CHALOIN-DUFAUC., SUN T-T., DHOUAILLYD. (1990). Cell Different.Develop. 32, 97-108. SUN T-T., EICHNERR., SHERMERA., COOPERD., NELSONWG., WEISS RA. (1984). In "Cancer cell', vol 1, The Iransformedphenolype,LevineA, Topp W, VandeWoudeG, WatsonJD, eds, ColdSpringHarbor,169-176.
(1) GANDRILLON O., JURDIC P., PAIN B.. DESBOIS C., MAD,JAR J.,l., MOSCOVICI M.G.. MOSCOVICI C. et SAMARUT d. (1989}. Cell 58, 115-121. {21 PAIN B., MELET F., JURDIC P. et SAMARUT J. (1990}. Tile New Biologist 2. 284-294. [31 MARCHAL S.. CASSAR-MALEK I., WRUTNIAI{ C.. CABELLO G. Molecular control of tile cell cycle ENS Lyon, 6-8 dulllel 1992
DOWN R E G U L A T I O N O F CDC2 E X P R E S S I O N IN T I l E DEVELOPING QUAIL NEURORETINA g l L L E T Germain, ESPANEL Xavier, STETTLER Olivier*, T A V I T I A N Bertrand*, BRUN Gilbert ENS Lyon, 46 allde d'halie 69364 Lyon cddex 07 *SHFJ,CEA, 4 place du G al Leclerc 91401 Orsay cdde.r In the d e v e l o p i n g quail neuroretina, n u m e r o u s cell divisions are observed before embryonic day E7 while no mitosis can be detected after day E8 suggesting that a stringent negative control of cell divisions occurs at this stage, ht an attempt to understand the molecular events i n v o l v e d in this g r o w t h arrest, cdc2 e x p r e s s i o n was a n a l y s e d in the d e v e l o p i n g quail n e u r o r e t i n a . We have observed, both at the m R N A and at the protein levels, a high e x p r e s s i o n of cdc2 before day E8 followed by a marked d e c r e a s e after day E9. This suggests a negative regulation of cdc2 expression at the transcriptional level. T h e amount of the Rb protein is also down regulated following the same kinetics as p 3 4 c d c 2 . The s y n c h r o n o u s decrease of p34 c d c 2 and Rb could be a major event leading to postmitotic n e u r o n a l precursors. In situ h y b r i d i z a t i o n e x p e r i m e n t s u s i n g a synthetic 3 5 S o l i g o n u c l e o t i d e as a probe, have c o n f i r m e d this n e g a t i v e control of cdc2 e x p r e s s i o n . An i n t e n s e and h o m o g e n e o u s labe~lling was observed in all cell layers before day E7 and no labelling could be detected after day E8. However, at day E7 the i n n e r n u c l e a r layer was still labelled s u g g e s t i n g that p o s t m i t o t i c a m a c r i n e n e u r o n a l p r e c u r s o r s still e x p r e s s cdc2. Therefore, in some cell populations of the developing n e u r o r e t i n a , p34 o d e 2 could be involved in the early steps of differentiation occuring immediately after the arrest of cell division.
HORMONAL CONTROL OF CELL-TO-CELL COlVlMUNICATION AND OF CONNEXIN GENE EXPRESSION IN THE THYROID. MUNARI-SILEM Yvonne(l), GUERRIER Annie(l), FROMAGET Catherine(2), KRUTOVSKIKH Vladimig3), EL AOUMARI Abdelhakim(2), GROS Daniel(2), ROUSSET Bernard(I) (ll INSERM U. 197, Facultdde MddecineA. Carrel,Lyon; (2) L.A. CNRS 179, Facultd des Sciencesde Luminy,Marseille;(3) InternationalAgencyfor Research Cancer,Lyon. Gap junctions (Gj) are constituted by the aggregation of many hydrophilic channels which mediate direct cell-to-cell communication. Gj are involved in metabolic coupling and in the control of cell growth and differentiation. In some tissues, the synthesis of Gj constituting protein: connexin (Cx) and the "functioning" (i.e. opening or closure) of the Gj channels seem to be under homlonal dependance. In thyroid, we attempted to determine 1) whether cultured thyroid ceils are able to reconstitute functional gap junctions, 2) whether channel gating is regulated by TSH, 3) whether Cx gene expression is regulated by TSH. The direct cell-to-cell cormrtunication was studied by microinjection of a fluore:;cent probe (Lucifer Yellow) in pig thyroid cells cultured for 1 to 8 days in the presence or the absence of TSH.The expression of Cx mRNA was analyzed by northern blot using eDNA probes for the two best studied Cx: Cx32 and Cx43. The content and the distribution of the two Cx were analyzed by immunofluorescence using polyclonal antipepdde antibodies for Cx32 and Cx43. The level of cell-to-cell communication remained high tot up to I0 days in cells cultured with TSH, while it rapidly decreased in cells cultured without TSH. Acute TSH treatment rapidly increased the communication between cells cultured in the absence of TSH; this effect was dose- and time-dependent. High amounts of the 1.6 kb Cx32 tmRNA were detected in freshly isolated thyrocytes (ITC); the level of this transcript decreased in cells cultured in the absence of TSH and remained high in the presence of TSH. ITC contained only trace amounts, of the 3 kb Cx43 transcript. Interestingly, the amount of this transcript increased in cells cultured in the presence of TSH. The amount of Cx43 protein detected by western blot or immuno-fluorescence was higher in cells cultured in the presence of TSH than in the absence of TSH. These results demonstrated that: 1) Cultured thyroid epithelial cells rapidly reconstitute functional Gj which appeared positively modulated by TSH. 2) .Thyroid cells which express very preferentially Cx32 "in situ", still express Cx32 and progressively express Cx43 in culture. 3) The expression of these two Cx was positively.modulated by TSH. In conclusion, TSH regulates both Gj gating and the expression of their constitutive proteins.
219
I N F L U E N C E OF T H E O N C O P R O T E I N v - e r b A ON Q U A I L MYOBLAST P R O L I F E R A T I O N A N D D I F F E R E N T I A T I O N .
D I F F E R E N T I A T I O N OF THE R A B B I T EMBRYO C O R N E A L E P I T H E L I U M : E X P R E S S I O N OF T H E A C I D I C KERATIN K12.
C A S S A R - M A L E K l,~abelle, MARCHAL S o p h i e , WRUTN1AK C h a n t a l , SAMARUT d a e q u e s * , CABELLO G 6 r a r d . U n i t 6 d e D l f f 6 r e n c l a t l o n C e l l u l a i r e et C r o l s s a n c e , 1NRA. P l a c e Viala, 3 4 0 6 0 MONTPELLIER C e d e x 1. * Lab. Biologic M o l 6 c u l a i r e et C e l l u l a l r e , C N R S U M R 4 9 , INRA E N S Lyon, 4 6 all6e tl'ltalle, 6 9 3 6 4 Lyon C~dex 0 7
CHALOIN-DUFAU Catherin~ 1, SUN Tung-Tien2 and DHOUAILLY Danielle1. 1- Laboratoire de Biologie de la Diffdrenciation Epithdliale. Universitd Joseph Fourier. CERMO. BP 53X. 38041 GRENOBLE. 2- Epithelial Biology Unit. Department of Dermatology. New York Universi~/ School of Medicine. NEW YORK.
N u m e r o u s d a t a e v i d e n c e t h a t t h e e x p r e s s i o n of t h e ve r b A o n e o g e n e I n h l b l t s t h e d i f f e r e n t i a t i o n of e r y t b r o l d cell p r e c u r s o r s (1) a n d t h e e x p r e s s i o n of s p e c i f i c e r y t h r o c y t l c g e n e s (2). It Is g e n e r a l l y a s s u m e d t h a t t h e v - e r b A p r o t e i n Is a b l e to a n t a g o n i z e t h e effects of t h e T 3 / n u c l e a r r e c e p t o r complexes on gene transcription. As we h a v e s h o w n t h a t T3 i n f l u e n c e s q u a i l m y o b l a s t p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n (3), we h a v e s t u d i e d t h e I n f l u e n c e of v - e r b A In o u r c u l t u r e model. We h a v e I n f e c t e d myoblasts with a recombinant retrovirus carrying the v-erbA o n c o g e n e . In t h e a b s e n c e of T3 ( a n d p r o b a b l y of r e t l n o i c acid), v - e r b A s i g n i f i c a n t l y I n c r e a s e s t h e p r o l i f e r a t i o n r a t e of myoblasts. Moreover. It e n h a n c e s accumulation of acetylcholine r e c e p t o r s a n d of m y o s i n h e a v y c h a i n , measured as differentiation markers. These results bring the first evidence that v-erbA could affect cell proliferation and differentiation I n d e p e n d e n t l y of T3 a n d r e t l n o l c acid. T h e y r a i s e also t h e following q u e s t i o n s : Is v - e r b A a b l e to s t i m u l a t e m y o b l a s t d i f f e r e n t i a t i o n ? A l t e r n a t i v e l y , d o e s It f a v o u r t h e e m e r g e n c e of m y o b l a s t s w i t h h i g h d i f f e r e n t i a t i o n p o t e n t i a l i t i e s , from a heterogencous myoblast population?
The K3/K12 keratins pair serves as biochemical marker for an advanced slage of "corneal-type" epithelial differenliation (Sun et al., 1984). Using an AK12 monoclonal antibody (Chaloin-Dufau et aL, 1990) highly specific for the acidic keratin K12, we have isolated a cDNA of this keralin from a ~.gll 1 rabbit corneal epithelium library. Sequential expression of the K12 keratin and of its mRNA was studied during embryonic d e v e l o p m e n t using bolh immunofluorescent slaining and in situ hybridization. The appearance of the protein and lhe activation of the corresponding gene are strictly synchronized: they appear first in Ihe suprabasal layer of central corneal epithelium at 17 days of gestation, and reach the basal epithelial layer six days later (23 days of gestation). Therefore, the regulation of the acidic keratin K12 gene appears to be primarily at the transcriptional level. CHALOIN-DUFAUC., SUN T-T., DHOUAILLYD. (1990). Cell Different.Develop. 32, 97-108. SUN T-T., EICHNERR., SHERMERA., COOPERD., NELSONWG., WEISS RA. (1984). In "Cancer cell', vol 1, The Iransformedphenolype,LevineA, Topp W, VandeWoudeG, WatsonJD, eds, ColdSpringHarbor,169-176.
(1) GANDRILLON O., JURDIC P., PAIN B.. DESBOIS C., MAD,JAR J.,l., MOSCOVICI M.G.. MOSCOVICI C. et SAMARUT d. (1989}. Cell 58, 115-121. {21 PAIN B., MELET F., JURDIC P. et SAMARUT J. (1990}. Tile New Biologist 2. 284-294. [31 MARCHAL S.. CASSAR-MALEK I., WRUTNIAI{ C.. CABELLO G. Molecular control of tile cell cycle ENS Lyon, 6-8 dulllel 1992
DOWN R E G U L A T I O N O F CDC2 E X P R E S S I O N IN T I l E DEVELOPING QUAIL NEURORETINA g l L L E T Germain, ESPANEL Xavier, STETTLER Olivier*, T A V I T I A N Bertrand*, BRUN Gilbert ENS Lyon, 46 allde d'halie 69364 Lyon cddex 07 *SHFJ,CEA, 4 place du G al Leclerc 91401 Orsay cdde.r In the d e v e l o p i n g quail neuroretina, n u m e r o u s cell divisions are observed before embryonic day E7 while no mitosis can be detected after day E8 suggesting that a stringent negative control of cell divisions occurs at this stage, ht an attempt to understand the molecular events i n v o l v e d in this g r o w t h arrest, cdc2 e x p r e s s i o n was a n a l y s e d in the d e v e l o p i n g quail n e u r o r e t i n a . We have observed, both at the m R N A and at the protein levels, a high e x p r e s s i o n of cdc2 before day E8 followed by a marked d e c r e a s e after day E9. This suggests a negative regulation of cdc2 expression at the transcriptional level. T h e amount of the Rb protein is also down regulated following the same kinetics as p 3 4 c d c 2 . The s y n c h r o n o u s decrease of p34 c d c 2 and Rb could be a major event leading to postmitotic n e u r o n a l precursors. In situ h y b r i d i z a t i o n e x p e r i m e n t s u s i n g a synthetic 3 5 S o l i g o n u c l e o t i d e as a probe, have c o n f i r m e d this n e g a t i v e control of cdc2 e x p r e s s i o n . An i n t e n s e and h o m o g e n e o u s labe~lling was observed in all cell layers before day E7 and no labelling could be detected after day E8. However, at day E7 the i n n e r n u c l e a r layer was still labelled s u g g e s t i n g that p o s t m i t o t i c a m a c r i n e n e u r o n a l p r e c u r s o r s still e x p r e s s cdc2. Therefore, in some cell populations of the developing n e u r o r e t i n a , p34 o d e 2 could be involved in the early steps of differentiation occuring immediately after the arrest of cell division.
HORMONAL CONTROL OF CELL-TO-CELL COlVlMUNICATION AND OF CONNEXIN GENE EXPRESSION IN THE THYROID. MUNARI-SILEM Yvonne(l), GUERRIER Annie(l), FROMAGET Catherine(2), KRUTOVSKIKH Vladimig3), EL AOUMARI Abdelhakim(2), GROS Daniel(2), ROUSSET Bernard(I) (ll INSERM U. 197, Facultdde MddecineA. Carrel,Lyon; (2) L.A. CNRS 179, Facultd des Sciencesde Luminy,Marseille;(3) InternationalAgencyfor Research Cancer,Lyon. Gap junctions (Gj) are constituted by the aggregation of many hydrophilic channels which mediate direct cell-to-cell communication. Gj are involved in metabolic coupling and in the control of cell growth and differentiation. In some tissues, the synthesis of Gj constituting protein: connexin (Cx) and the "functioning" (i.e. opening or closure) of the Gj channels seem to be under homlonal dependance. In thyroid, we attempted to determine 1) whether cultured thyroid ceils are able to reconstitute functional gap junctions, 2) whether channel gating is regulated by TSH, 3) whether Cx gene expression is regulated by TSH. The direct cell-to-cell cormrtunication was studied by microinjection of a fluore:;cent probe (Lucifer Yellow) in pig thyroid cells cultured for 1 to 8 days in the presence or the absence of TSH.The expression of Cx mRNA was analyzed by northern blot using eDNA probes for the two best studied Cx: Cx32 and Cx43. The content and the distribution of the two Cx were analyzed by immunofluorescence using polyclonal antipepdde antibodies for Cx32 and Cx43. The level of cell-to-cell communication remained high tot up to I0 days in cells cultured with TSH, while it rapidly decreased in cells cultured without TSH. Acute TSH treatment rapidly increased the communication between cells cultured in the absence of TSH; this effect was dose- and time-dependent. High amounts of the 1.6 kb Cx32 tmRNA were detected in freshly isolated thyrocytes (ITC); the level of this transcript decreased in cells cultured in the absence of TSH and remained high in the presence of TSH. ITC contained only trace amounts, of the 3 kb Cx43 transcript. Interestingly, the amount of this transcript increased in cells cultured in the presence of TSH. The amount of Cx43 protein detected by western blot or immuno-fluorescence was higher in cells cultured in the presence of TSH than in the absence of TSH. These results demonstrated that: 1) Cultured thyroid epithelial cells rapidly reconstitute functional Gj which appeared positively modulated by TSH. 2) .Thyroid cells which express very preferentially Cx32 "in situ", still express Cx32 and progressively express Cx43 in culture. 3) The expression of these two Cx was positively.modulated by TSH. In conclusion, TSH regulates both Gj gating and the expression of their constitutive proteins.