Hypocalcemia decreases the early and late responses to epidermal growth factor in rat hepatocytes

Hypocalcemia Decreases the Early and Late Responses to Epidermal Growth Factor in Rat Hepatocytes MARC B I L O D E A U , 1 Sl~BASTIEN J. P R O V E N C H E R , 1'2 STI~PHANE Nt~RON, ~'2 P I E R R E H A D D A D , 2 SYLVIE VALLII~RES, 1 AND MARIELLE GASCON-BARRIO,1'2

E x t r e m e v a r i a t i o n s in extracellular Ca 2÷ c o n c e n t r a t i o n s ([Ca2+]e) m o d i f y t h e signaling g e n e r a t e d by m a n y h o r m o n e s a n d g r o w t h factors. H o w e v e r , t h e i n f l u e n c e o f p h y s i o l o g i c a l c h a n g e s in [Ca2+]e o n t h e r e s p o n s e to h e p a t i c m i t o g e n s r e m a i n s largely u n k n o w n . To s t u d y the i n f l u e n c e o f [Ca2+]e o n t h e r e s p o n s e to e p i d e r m a l g r o w t h factor (EGF), h e p a t o c y t e s f r o m n o r m a l rat livers w e r e equilibrated in vitro at [Ca2+]e similar to t h o s e obs e r v e d in n o r m o c a l c e m i a or h y p o c a l c e m i a . To f u r t h e r i n v e s t i g a t e t h e effect o f h y p o c a l c e m i a in vivo, hepatocytes w e r e o b t a i n e d f r o m c h r o n i c a l l y h y p o c a l c e m i c rats a n d k e p t in vitro at t h e [Ca2÷]~ p r e v a i l i n g in vivo. Intracellular Ca 2÷ c o n c e n t r a t i o n s ([Ca2÷]i) a n d D N A s y n t h e s i s w e r e e v a l u a t e d after i n c r e a s i n g d o s e s o f EGF. [Ca2÷]e s t r o n g l y i n f l u e n c e d t h e [Ca2÷]i r e s p o n s e to EGF w i t h significantly s m a l l e r [Ca2+]i i n c r e a s e s in h e p a t o c y t e s o f norm a l rats k e p t in l o w [Ca2+]e c o m p a r e d w i t h t h o s e k e p t in n o r m a l [Ca2+]e. In h y p o c a l c e m i c rat h e p a t o c y t e s , the r e s p o n s e w a s f u r t h e r d e c r e a s e d a n d f o u n d to be significantly l o w e r t h a n that o b t a i n e d in c o n t r o l cells k e p t in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca2÷]e. In norm a l [Ca2÷]~, t h e EGF-induced i n c r e a s e s in [Ca2+]i w e r e a b o l i s h e d by i n h i b i t i n g EGF r e c e p t o r a u t o p h o s p h o r y l a t i o n a n d by b l o c k i n g c a l c i u m c h a n n e l s . L o w in vitro [Ca2+]e significantly d a m p e n e d t h e E G F - m e d i a t e d D N A s y n t h e s i s in n o r m a l rat h e p a t o c y t e s but h y p o c a l c e m i a in vivo f u r t h e r r e d u c e d t h e proliferative r e s p o n s e comp a r e d w i t h that o b t a i n e d in c o n t r o l rat h e p a t o c y t e s m a i n t a i n e d in n o r m a l , or l o w [Ca2÷]~. F u r t h e r m o r e , t h e b l u n t e d r e s p o n s e s in [Ca2÷]i m o b i l i z a t i o n a n d D N A synthesis a s s o c i a t e d w i t h h y p o c a l c e m i a c o u l d n o t be overAbbreviations: TGF, transforming growth factor; EGF, epidermal growth factor; HGF, hepatocyte growth factor; [Ca~+]i,intracellular calcium concentration; [Ca2+]e, extracellular calcium concentration. From the 1Centre de recherche clinique Andr~-Viallet, HSpital Saint-Luc, and 2D6partement de pharmacologie, Facult6 de m~decine, Universit6 de Montreal, Montreal, Canada. Received May 24, 1994; accepted December 19, 1994. Supported by the Medical Research Council of Canada, Ottawa, Ontario, Canada. Marc Bilodeau and S~bastien J. Provencher are, respectively, recipients of postdoctoral Fellowships and of Studentship Awards from the Canadian Liver Foundation; Pierre Haddad is the recipient of a chercheur-boursier from the ~Fonds de la recherche en sant~ du Qu6bec>. Address reprint requests to: Marielle Gascon-Barr6, PhD, Centre de recherthe clinique Andre-Viallet, H6pital Saint-Luc, 264 Ren~-L6vesque Blvd East, Montreal, Quebec H2X 1P1, Canada. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2106-001553.00/0

c o m e by i n c r e a s i n g c o n c e n t r a t i o n s of EGF n o r by norm a l i z a t i o n o f [Ca2+]e in vitro. T h e s e data d e m o n s t r a t e that [Ca2*]e w i t h i n t h e p h y s i o l o g i c a l c o n c e n t r a t i o n r a n g e c a n strongly i n f l u e n c e t h e h e p a t o c y t e r e s p o n s e to EGF. At [Ca2*]e c o m p a r a b l e to that of h y p o c a l c e m i a in vivo, o u r data p o i n t to the a p p e a r a n c e o f a p h e n o m e n o n of h e p a t o c e l l u l a r r e s i s t a n c e to t h e early (increases in [Ca2*]i) a n d late (DNA synthesis) cellular r e s p o n s e s to EGF. (HEPATOLOGY1995;21:1576.1584.)

The liver has the extraordinary capacity to modulate its mass to meet the metabolic activities that bear on its functions. In vivo, this is well illustrated by the regenerative process that takes place after either partial hepatectomy or necrotic insults leading to liver cells losses. 1 Clinically, the regenerative capacity of the liver is also well illustrated by the adaptive response of transplanted livers, which, if needed, will increase in size to fit the recipients' size and meet the metabolic demands. 2 The regenerative phenomenon is due to the triggering of cellular events capable of initiating, in some liver cells, a reentry into the cell cycle. These activated cells will then allow the liver to grow until it has reached the critical mass necessary to meet its function. In vitro studies have clearly shown that hepatocytes are able to proliferate in response to well-defined conditions as exemplified by the response observed after stimulation by several growth factors such as transforming growth factor alpha (TGF-a) or epidermal growth factor (EGF) (acting at the same receptor), hepatocyte growth factor (HGF), and acidic fibroblast growth factor. These are considered as true liver mitogens. 3 In addition, several co-mitogens such as insulin, glucagon, vasopressin, and noradrenaline, as well as monovalent and divalent cations, have been identified in vitro and considered to be necessary to sustain the compensatory growth process. 4-6 By contrast, transforming growth factor beta (TGF-/3) has been proposed as responsible for mediating the arrest in cell growth 7 by acting in a paracrine manner on hepatocytes. Mitogenic stimuli induced by growth factors depend on complex signal transduction mechanisms that are initiated by their specific binding to plasma membrane receptor. The early increase in intracellular calcium concentration ([Ca2+]~) is now recognized as a necessary

1576

HEPATOLOGYVol. 21, No. 6, 1995

BILODEAU ET AL

1577

TABLE 1. E x p e r i m e n t a l D e s i g n Animals Extracellular CA~+

Normal Rats

In vivo serum ionized calcium (mmol/L)

D Deficient R a t s

1.26 ± 0.01

0.80 _+0.03***

]

I

In vitro media ionized calcium (mmol/L)

0.84 + 0.02

I

]

1.23 ± 0.01"**

I

0.82 ± 0.02

I

1.25 ± 0.01"**

NOTE. Ionized calcium concentrations observed in vivo and in vitro in the solutions or media bathing the hepatocyte primary culture used during the experimental periods; n = 7 in each group. Data are presented as means _+ SEM. Statistically significant differences between group means were analyzed by the Student's t-test. ***P < .0001. s t e p of t h e physiological r e s p o n s e c h a r a c t e r i z e d b y a c a s c a d e of cellular s u b s t r a t e a c t i v a t i o n t h r o u g h t h e p a r t i c i p a t i o n o f k i n a s e s a n d p h o s p h a t a s e s t h a t link t h e m i t o g e n i c cell s u r f a c e signal to t h e n e c e s s a r y n u c l e a r actions i n v o l v e d in t h e p r o l i f e r a t i v e r e s p o n s e . L a t e l y , s t u d i e s c a r r i e d out in o u r l a b o r a t o r y h a v e clearly s h o w n t h a t h y p o c a l c e m i a a n d v i t a m i n D (D) deficiency a r e a c c o m p a n i e d b y modifications in t h e r e s t i n g h e p a tocyte [Ca2+]is as well as in t h e h e p a t i c r e g e n e r a t i v e process. 911 F u r t h e r s t u d i e s w e r e able to u n c o v e r t h a t h y p o c a l c e m i a , i n vivo, is a c c o m p a n i e d b y a significant d e c r e a s e , n o t only in E G F r e c e p t o r density, b u t also in E G F r e c e p t o r a u t o p h o s p h o r y l a t i o n , a n d p r o t e i n tyrosine k i n a s e activity. 12 T h e s e o b s e r v a t i o n s t h u s r a i s e d t h e h y p o t h e s i s t h a t t h e defective r e g e n e r a t i o n p h e n o m e n o n t h a t a c c o m p a n i e s h y p o c a l c e m i a is d u e to defect(s) in t h e m i t o g e n i c s i g n a l i n g s y s t e m of t h e h e p a t o c y t e . A l t h o u g h s e v e r a l s t u d i e s h a v e i n v e s t i g a t e d t h e role of extreme extracellular calcium concentrations ([Ca2+]e) ( m o s t often well outside t h e physiological r a n g e ) on s e v e r a l signal t r a n s d u c t i o n p a r a m e t e r s in h e p a t o c y t e s , no s t u d i e s h a v e y e t i n v e s t i g a t e d t h e influence of [ C a 2 + ] e c o m p a t i b l e w i t h s t a t e s of h y p o c a l c e m i a or n o r m o c a l c e m i a i n v i v o on t h e r e s p o n s e of h e p a t o c y t e s to t h e powerful hepatic mitogen EGF. T h e c u r r e n t s t u d i e s w e r e t h e r e f o r e a i m e d a t investigating, i n vitro, t h e effect of physiological c o n c e n t r a tions of e x t r a c e l l u l a r c a l c i u m on t h e liver r e s p o n s e to EGF, taking hepatocytes obtained from either normal, or h y p o c a l c e m i c r a t livers as e x p e r i m e n t a l p a r a d i g m . We now report that extracellular calcium concentrations c o m p a r a b l e to t h o s e o b s e r v e d in h y p o c a l c e m i a i n v i v o significantly influence t h e h e p a t i c r e s p o n s e to E G F , as i l l u s t r a t e d b y a d e c r e a s e in t h e E G F - s t i m u l a t e d [Ca2+]i r e s p o n s e as well as b y a d a m p e n i n g of t h e h e p a t o c y t e D N A s y n t h e s i s , a p h e n o m e n o n t h a t could not be o v e r c o m e b y i n c r e a s i n g c o n c e n t r a t i o n s of E G F n o r b y n o r m a l i z a t i o n of [Ca2+]e i n vitro. MATERIALS

AND

METHODS

Experimental Design To study the influence of the [Ca2*]e on the response to EGF, hepatocytes were isolated from normal rat livers and equilibrated in vitro in culture medium adjusted at ionized calcium concentrations similar to those observed in either normocalcemia (~1.25 mmol/L Ca 2+) or hypocalcemia ( - 0 . 8 mmol/L Ca 2+) in vivo (Table 1). To further investigate the

effect of hypocalcemia in vivo on the EGF response, hepatocytes were also obtained from chronically hypocalcemic rats and kept in vitro at an [Ca2+]e comparable to the prevailing ionized calcium in vivo ( - 0 . 8 mmol/L Ca~÷). The cellular response to EGF was investigated after increasing concentrations of EGF by measuring the [Ca2+]i response as marker of the early signal transduction, and DNA synthesis as indicator of its mitogenic potential.

Animals Male Sprague-Dawley rats (Charles River Canada, Ltd., St. Constant, Quebec, Canada) were used for all experiments. They were fed a regular rat chow diet and tap water ad libitum, and kept in a controlled environment with a 12hour light-dark cycle. To evaluate the influence of chronic hypocalcemia on the response to EGF, a group of animals was subjected to a functional calcium deprivation through vitamin D depletion as previously described. 9,1~ This procedure does not influence liver function. 9'14 Moreover, somatic growth is not severely impaired because the diet provides adequate amounts of minerals and other nutrients but leads to hypocalcemia due to the progressive depletion in vitamin D as evidenced by circulating 25-hydroxyvitamin D concentrations near undetectable levels. 9 Concentrations of ionized calcium in whole blood, and in all culture and experimental solutions, were measured with a ICA2 ionized calcium analyzer (Radiometer, Copenhagen, Denmark). All animals were treated according to the standards of ethics for animal experimentation of the Canadian Council of Animal Care. All protocols were approved by the ]ocal animal ethics committee.

Response of Hepatoeytes to E G F Materials. Chemical reagents were obtained from the following suppliers: EGF, verapamil, dexamethasone, pyruvate, fibronectin (Sigma Chemical Co., St Louis, MO), William E medium, tyrphostin, penicillin G, streptomycin (Gibco BRL, Burlington, Ont., Canada), FURA 2-AM (Molecular Probes Inc., Eugene, OR), insulin (Eli Lilly and Company, Indianapolis, IN), and [methyl-3H]thymidine (ICN Biomedicals Canada Ltd, Mississauga, Ontario, Canada). Cell Isolation. Hepatocytes were obtained from livers of nonfasting rats using a two-step collagenase perfusion as previously reported. 15 The freshly isolated hepatocytes were equilibrated in William E medium containing ionized calcium concentrations similar to those observed in normocalcemia or hypocalcemia in vivo as indicated in Table 1. Cell yield and viability (evaluated by the Trypan blue exclusion test) were found to be similar in all groups. Hepatocyte viability averaged 95% and 92% in cells obtained from normal and hypocalcemic rat livers, respectively.

1578

BILODEAU ET AL

Intracellular Calcium Measurements. Hepatocytes were p l a t e d (3.5 to 5 × 10 ~ cells/mL) onto collagen-coated coverslips in W i l l i a m s E m e d i u m containing 25 mmol/L b i c a r b o n a t e a n d 1% bovine s e r u m albumin, p H 7.4, 37°C in 5% C Q atmosphere. After 60 m i n u t e s of culture, cells were loaded for 30 m i n u t e s a t 20°C w i t h 2.5 #mol/L F U R A 2-AM in bicarbonatefree W i l l i a m s E m e d i u m s u p p l e m e n t e d w i t h 2.5% fetal bovine s e r u m a n d 1% bovine s e r u m albumin. Dye-loaded cells were t r a n s f e r r e d into a 100-#L plastic c h a m b e r to t h e stage of an i n v e r t e d microscope (Nikon Diaphot, Nikon Corp., Tokyo, J a p a n ) equipped for epifluorescence m e a s u r e m e n t . Cells were perfused at a r a t e of 2 m L / m i n w i t h a K r e b s - H e n s e l e i t buffer e q u i l i b r a t e d with O2:CO2 (95:5, vol:vol) at 37°C. Fluorescence signals were obtained from hepatocyte couplets w i t h a SPEX model CMIT-11I d u a l excitation spectrofluorometer (Rayonics Scientific Inc., St L a u r e n t , Quebec). Excitation w a v e l e n g t h s were 350 a n d 380 nm, a n d fluorescence emission was m e a s u r e d at 505 nm. I n t r a c e l l u l a r dye calibration was performed in situ by perfusion of 10 #mol/L ionomycin in a solution containing 4 mmol/L ethylene glycol t e t r a - a c e t i c acid (Rmln) or 4 mmol/L CaC]2 (Rm~x). After correction for s a m p l e autofluorescence, signal ratios (F3~0/F3so) were t r a n s f o r m e d into [Ca2+]i.l~ I n t r a c e l l u l a r dye s p e c t r a a n d loading capacities were found to be e q u i v a l e n t in all groups as a l r e a d y reported, s All t e s t compounds were perfused in K r e b s - H e n s e l e i t solution a d j u s t e d a t 1.25 mmol/L or 0.8 mmol/L [Ca2+] e. A d o s e response curve was performed u s i n g E G F at concentrations of 5 to 100 ng/mL. To i n v e s t i g a t e t h e m e c h a n i s m s involved in the [Ca2+]i response, tyrphostin, an inhibitor of t h e E G F receptor autophosphorylation, was used a t a concentration of 247 #mol/L, a n d v e r a p a m i l , a calcium channel blocker active on r e c e p t o r - o p e r a t e d calcium channels in hepatocytes, 17 was used at a concentration of 20 #mol/L. The i m p o r t a n c e of e x t r a c e l l u l a r Ca 2+ ions in the [Ca2+]i response to E G F was e v a l u a t e d by a d d i n g 500 #mol/L Mn to the perfusion buffer of FURA-2 A M - l o a d e d h e p a t o c y t e s obtained from hypocalcemic a n d normocalcemic r a t livers, a n d k e p t in vitro a t e i t h e r 1.25 or 0.8 mmol/L Ca 2+. The Mn 2÷m e d i a t e d fluorescence quenching of the FURA-2 signal was t h e n m o n i t o r e d at t h e excitation w a v e l e n g t h s of 334 a n d 366 nm. The s i m u l t a n e o u s a n d p a r a l l e l loss of fluorescence observed a t both w a v e l e n g t h s is indicative of M n 2+ e n t r y t h r o u g h receptor-operated, v o l t a g e - i n d e p e n d e n t Ca 2+ channel. is The cellular e n t r y of Mn 2÷ m e d i a t e d by E G F (50 ng/ mL) application was e v a l u a t e d by calculating t h e slopes induced by t h e loss of t h e fluorescence signal secondary to Mn 2+ b i n d i n g to the FURA-2 probe. DNA Synthesis. F r e s h l y isolated hepatocytes r e s u s p e n d e d in W i l l i a m s E m e d i u m containing e i t h e r 1.25 or 0.8 mmol/L Ca 2+ were s u p p l e m e n t e d with 150 #g/L insulin, 4 #mol/L d e x a m e t h a s o n e , 0.25 #mol/L p y r u v a t e , 100 U/mL penicillin G, a n d 100 #g/mL streptomycin. They were p l a t e d in 35-mm d i a m e t e r dishes coated w i t h 3 #g/cm 2 fibronectin at a cell d e n s i t y of 2.5 × 10 ~ p e r dish. Cells were i n c u b a t e d at 37°C u n d e r CO2:air (5:95, v:v). After 3 hours, t h e m e d i u m was changed to fresh W i l l i a m s E m e d i u m containing 5 ng/L insulin, 0.25 #mol/L p y r u v a t e , 100 U/L penicillin, a n d 100 #g/ m L streptomycin. E G F was a d d e d to the b a s a l m e d i u m in concentrations of 1 to 100 ng/mL. [3H]thymidine incorporation was used to e v a l u a t e DNA synthesis. Twenty-four hours after plating, 1 #Ci/mL [methyl3H]thymidine (spec. act., 70 to 90 Ci/mmo]) was a d d e d to fresh m e d i u m . D N A was p r e c i p i t a t e d 24 hours l a t e r for t h e

HEPATOLOGYJune 1995 p<0.0001 , r--- p<0.0001

NS

250

'i

200

".~ ,.~

150

+ e..I

t,...t

l(w)

511

Normal

Low

Low

Extracellular ionized calcium

FIG. 1. Basal [Ca2÷]iin freshly isolated hepatocytes isolated from livers of normal rats kept in short-term primary culture (1 hour) containing normal (1.25 mmol/L) • (n = 28), or low (0.8 mmo]/L) [] (n = 28) extracellular ionized calcium, or from hypocalcemic D-deficient rats kept in primary culture containing 0.8 mmol/L extracellular ionized calcium [] (n = 16). Data are presented as means _+ SEM and represent the number of hepatocyte preparations evaluated in seven animals in each group. Statistically significant differences between group means were analyzed by a one-way ANOVA (P < .0001) with individual contrasts between group means evaluated by the Tukey's test (bracketed values). d e t e r m i n a t i o n of [3H]thymidine incorporation as described p r e v i o u s l y ) 9 Hepatocyte v i a b i l i t y was e v a l u a t e d at the time of cell h a r v e s t i n g by expressing t h e i n t r a : e x t r a c e l l u l a r lactic d e h y d r o g e n a s e concentrations ratio. E x t r a c e l l u l a r lactic deh y d r o g e n a s e was m e a s u r e d in culture m e d i u m while i n t r a cellular lactic d e h y d r o g e n a s e was m e a s u r e d from the s u p e r n a t a n t of w a s h e d p e r m e a b i l i z e d (Triton X-100, 0.2%) hepatocytes. Lactic d e h y d r o g e n a s e was a n a l y z e d using an a u t o m a t e d m u l t i a n a l y z e r s y s t e m in our hospital clinical bioc h e m i s t r y laboratory. P r o t e i n concentration was m e a s u r e d according to Bradford et al 2° u s i n g crystalline bovine s e r u m a l b u m i n as s t a n d a r d .

Statistical Analysis Results a r e expressed as m e a n s _+ SEM. S t a t i s t i c a l l y significant differences between group m e a n s were e v a l u a t e d by a n a l y s i s of variance, or by the S t u d e n t ' s t-test as indicated in t h e table a n d figure legends. RESULTS

Influence o f E x t r a c e l l u l a r C a l c i u m on the [Ca2+]i R e s p o n s e to E G F A s i l l u s t r a t e d i n F i g . 1, r e s t i n g [Ca2÷]~ w a s n o t sign i f i c a n t l y i n f l u e n c e d b y [Ca2+]e i n n o r m a l r a t h e p a t o -

HEPATOLOGY Vol. 21, No. 6, 1995

BILODEAU ET AL

ited a dampened [Ca2+] i response to EGF (Fig. 2B). This dampening effect was strongly accentuated in preparations of hepatocytes obtained from chronically hypocalcemic rats (Fig. 2C) where hardly any significant response to EGF application could be observed. Figure 3 presents the d o s e - r e s p o n s e curves to EGF in the three groups studied. Extracellular Ca 2+ significantly influenced the [Ca2÷]i response to EGF over the dose range studied (P < .0001). In normal rat hepatocytes, peak [Ca2+]i was achieved at 50 ng/mL EGF in both normal and low [Ca2+]e (P < .03). However, at [Ca2+] e comparable to that of hypocalcemia in vivo, the [Ca2+]i response observed at the optimal EGF concentration was significantly decreased (P < .04). In hepatocytes obtained from hypocalcemic rat livers, the response was further decreased and found to be significantly different from that of normal rat hepato-

750 A

Normal

~'xA

[Ca2+l~1.25mM

500 • ..

250

i..F

..........

....

....

I

,

I

O

,

I

50

,

I

100

,

150

I

,

200

.....

i

l

250

300

750 B

Normal [Ca2+]e0.8 mM

,d

E J +-=

500

250 I

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I

0

50

10O

,

I

,

150

I

I

,

200

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i

250

1579

,

300 500

750 D deficient [Ca2+]¢0.8 mM

C

÷"~

400

%

500

3OO

T 250 I

0

,

I

50

,

I

i

i

100

150

200

,

I

250

,

I

,

•~

200

300

TIME (seconds) FIG. 2. Influence of the extracellular ionized calcium concentration on the p a t t e r n of [Ca2+]~ response after exposure to EGF. Tracings represent typical [Ca2+]i responses obtained during the continuous perfusion of 50 ng/mL EGF. Hepatocyte couplets were obtained from normal rat livers and maintained in vitro at an extracellular Ca 2+ concentration of 1.25 mmol/L (A), or 0.8 mmol/L (B), or from hypocalcemic D - d e f i c i e n t rat livers and maintained in vitro at an extracellular Ca 2+ concentration of 0.8 mmol/L (C). Hypocalcemic D deficient rat hypatocytes maintained in vitro at an extracellu]ar Ca e÷ concentration of 1.25 mmol/L exhibited responses similar to those maintained in 0.8 mmol/L Ca 2÷ (see quantitative data in Fig. 3). T indicates the beginning of EGF application.

cytes with concentrations of 237.9 _+ 11.7 and 231.0 + 10.5 nmol/L at 1.25 and 0.8 mmol/L extrace]lular Ca 2+, respectively. However', in hepatocytes obtained from hypocalcemic rats, resting [Ca2÷]i was found to be significantly lower (136.3 _+ 12.3 nmoUL) than that observed in normal rat hepatocytes kept either in normal (P < .0001), or low [Ca2+]o (P < .0001). Figures 2 and 3 present the [Ca2+]i response to EGF observed in hepatocytes obtained from normal as well as from hypocalcemic rat livers. As illustrated in the typical [Ca2+]i traces presented in Fig. 2, extracellular Ca 2+ clearly influenced the pattern of response to EGF. In comparison with normal conditions (Fig. 2A), normal rat hepatocyte preparations kept in low [Ca2+]~ exhib-

"O

•~

..............

100

0.~ 0

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I

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I

25

50

75

100

EGF

(ng/mi)

FIG. 3. Effect of the extracellular ionized calcium concentration on the [Ca2*]i response after exposure to increasing EGF concentrations. Hepatocyte couplets were obtained from normal rat livers and maintained in vitro at an extracellular Ca 2+ concentration of 1.25 (© C) (n = 7), or 0.8 mmol/L ( O - - 0 ) (n = 7), or from hypocalcemic D-deficient rat livers maintained at an extracellular Ca 2+ concentration of 0.8 mmol/L (11 - - II) (n = 7), or 1.25 mmoUL ([3 - - ½) (n = 2). EGF was perfused at concentrations varying from 5 to 100 ng/mL. Data represent the means _+ SEM for the peak [Ca2+]~ obtained at each EGF concentration (peak [Ca2+]i--paired basal [Ca~+]i values in each hepatecyte preparation). Statistically significant differences between group means were analyzed by analysis of variance taking the paired basal [Ca2% as co-variable of the response. Effect of EGF on [Ca2+]: P < .0001; effect of low extracel]ular calcium on the [Ca2+]i in normal rat hepatocytes: P < .04; effect of hypoca]cemia in vivo and 0.8 mmoUL [Ca2+]e in vitro in comparison with normal rat hepatocytes kept at 1.25 mmol/L Ca~+: P < .0002, or kept at 0.8 mmol/L Ca2*: P < .0001; effect of hypocalcemia in vivo and 1.25 mmoUL [Ca2+]~ in vitro in comparison with normal rat hepatocytes kept at 1.25 mmol/L Ca2+: P < .0001, or kept at 0.8 mmol/L Ca2+: P < .0001; effect of [Ca2+]e in hypocalcemic rat hypatocytes: NS. In normal hepatocytes, maximum [Ca2+]i was achieved at the EGF dose of 50 ng/mL P < .03; in hepatocytes of hypocalcemic rats, no significant effect was found in relation to the dose of EGF applied.

1580

BILODEAU ET AL 300 -

HEPATOLOGY J u n e 1995

1.25 mM Ca2+

0.8 mM Ca2+

250 O

200 150.

=

100-

basal value

50-

EGF Tyr Vera

+

+ ÷

+ +

+

+ +

+ +

FIG. 4. Tyrphostin- or verapamil-mediated inhibition of the EGFstimulated [Ca2+]~ response. Hepatocyte couplets were obtained from normal rat livers and maintained in vitro at an extracellular Ca 2+ concentration of 1.25 (normal) or 0.8 mmol/L (low). EGF was perfused at 50 ng/mL (8.2 nmol/L) in the presence of tyrphostin (247 #mol/L), or verapamil (20 #mol/L). Data (means _+ SEM) represent the EGFmediated increase in [Ca2+]i over paired unstimulated values for each condition. • EGF stimulation alone, [] EGF + tyrphostin, or [] EGF + verapamil. At 1.25 mmoFL Ca 2+, EGF stimulation (n = 8), inhibition by tyrphostin (n = 4), verapamil (n = 3); at 0.8 mmol/L Ca 2÷, EGF stimulation (n = 7), and n = 6 for each of the inhibitors. Statistically significant differences between group means were evaluated for each condition by the paired Student's t-test between the values observed under resting conditions (basal values) and the values observed in the presence of EGF alone or EGF and tyrphestin or verapamil. ***P < .003; *P < .05.

The importance of the [Ca2+]e on the response to EGF using Mn as indicator is presented in Fig. 5. In normocalcemic rat hepatocytes, the EGF-mediated Mn 2+ entry was influenced by [Ca2+]o (Fig. 5A) with slopes of FURA-2 quenching evaluated at -0.1707 _+ 0.0347 in normal [Ca2÷]e conditions but at -0.1594 + 0.0348 when [Ca2+]e was decreased to 0.8 mmoFL (P < .01 for paired hepatocyte preparations obtained from each rat). Hepatocytes obtained from hypocalcemic rats (Fig. 5B) exhibited a significantly reduced Mn2+-mediated quenching of the FURA-2 signal after EGF stimulation (P < .05), but [Ca2+] e did not influence Mn 2+ entry with slopes o f - 0 . 0 7 8 6 _+ 0.0305 at 1.25 mmo]/L [Ca2+] e and of -0.0710 _+ 0.0092 at 0.8 mmol/L [Ca2÷]~ (NS). I n f l u e n c e o f E x t r a c e l l u l a r C a l c i u m on the EGFStimulated DNA Synthesis

Figures 6 and 7 present the proliferative response to EGF in hepatocytes isolated from normal as well as from hypocalcemic rat livers. As illustrated in Fig. 6,

NORMOCALCEMICRAT

100.

80-

.

............

.............................. i ..........

.

60Z

cytes maintained in either normal (P < .001), or low [Ca2÷]~ (P < .002). Moreover, statistical analysis showed that hepatocytes obtained from hypocalcemic rat livers did not exhibit any significant dose-dependent increase in [Cae+]i in response to EGF stimulation. To evaluate whether the low [Ca2+]~ response observed in hepatocytes obtained from hypocalcemic rats livers was due to the in vivo conditioning effect of hypocalcemia, primary cultures ofhepatocytes from hypocalcemic rats were equilibrated for 1 hour in normal [Ca2÷]~ and stimulated with EGF in the presence of 1.25 mmol/ L [Ca2+]~. Normalization of [Ca2+]~ in vitro did not reverse the blunted [Ca2÷]~ response induced by the hypocalcemic state as illustrated in Fig. 3. Figure 4 presents the effect of tyrphostin and of verapamil on the [Ca2+]~ response to EGF in hepatocytes obtained in a subset of normal rat livers. In normal [Ca2÷]~, blocking the EGF receptor autophosphorylation with tyrphostin totally abolished the EGF-mediated increases in [Ca2+]~ with peak [Ca2+]i representing only +1.5 _+ 3.9% of those observed in the absence of EGF (paired basal values for each hepatocyte preparation) whereas verapami] blunted the [Ca2+]~ response by 85% when compared with that observed in the presence of EGF alone. In low [Ca2÷]~, the EGF-mediated increase in [Ca2+]i was not, however, significantly inhibited bv either tvrphostin or verapamil.

M"2"

A

}

0.8 mM

o: :: =

=:

== i

2

m.

b-

B

u"~

HYPOCALCEMICRAT

loo-~. 80-

0.8 mM Ca 2+

601

....~125 mM C ~ ÷

4oi. 20

iiiiiii~iIiIiiIiZiiIi-IiIi':~iI.:,'iIiIiiIiIiIiii!iili~i iiiiiii!iiiiiiiii.i.ii.i.i.iiiii::,

......

5~""i

- 1~i...;

1,~01 "'1

" 2_.~";

2~"~

TIME (SECONDS)

FIG. 5. Representative traces of the effect of [Ca2+]e on the Mn 2+ -mediated fluorescence quenching of the FURA-2 signal monitored at the excitation wavelength of 334 and 366 n m (traces shown were evaluated at 334 nm). Hepatocytes were isolated from normocalcemic (A) or hypocalcemic (B) rat livers and maintained for 1 hour in primary culture at 1.25 or 0.8 mmol/L [Ca2+]e. Extracellular Mn was added at a concentration of 500 # m as indicated by the arrows. Application of EGF is indicated by the arrows, and the receptor-operated voltage-independent Ca 2+ channel were blocked by Ni 2+ as indicated. In normocalcemic rats, n = 3 rats with two to three hepatocyte preparations/animal at each [Ca2+]e level. In hypocalcemic rats, n = 2 rats with two hepatocyte preparations/animal at each [Ca2+]e level.

HEPATOLOGY

o

Vol. 21, No. 6, 1995

BILODEAU

O

500

~ ~.

300

................. I ...................... ~

200

I

t

r~

~ m

o

1581

DISCUSSION

600

~

ET AL

I

[

I

I

25

50

75

100

EGF (ng/ml) FIG. 6. Effect of t h e e x t r a c e l l u l a r ionized c a l c i u m c o n c e n t r a t i o n on [3H]thymidine i n c o r p o r a t i o n a f t e r e x p o s u r e to i n c r e a s i n g E G F c o n c e n t r a t i o n s . H e p a t o c y t e s w e r e o b t a i n e d f r o m n o r m a l r a t livers a n d m a i n t a i n e d in p r i m a r y c u l t u r e at a n e x t r a c e l l u l a r Ca 2+ concent r a t i o n s of 1.25 (© ©) (n = 4), or 0.8 m m o l / L (O - - O) (n = 4), or f r o m h y p o c a l c e m i c D - d e f i c i e n t r a t livers m a i n t a i n e d a t 0.8 m m o l / L e x t r a c e l l u l a r Ca 2+ (ll - - II) (n = 7). C u l t u r e m e d i a w e r e s u p p l e m e n t e d w i t h E G F at c o n c e n t r a t i o n s r a n g i n g f r o m 1 to 100 n g / m L , a n d all d e t e r m i n a t i o n s w e r e m a d e in d u p l i c a t e dishes. D a t a r e p r e s e n t t h e m e a n s _+ S E M for e a c h of t h e conditions u s e d . S t a t i s t i c a l l y s i g n i f i c a n t differences b e t w e e n g r o u p m e a n s w e r e a n a l y z e d b y a n a l y sis of co-variance t a k i n g t h e r e s p o n s e in a b s e n c e of E G F as co-variable. Effect of E G F on [3H]thymidine incorporation, P < .00005; effect of low e x t r a c e l l u l a r c a l c i u m on [3H]thymidine i n c o r p o r a t i o n in norm a l r a t s , P < .004; effect of h y p o c a l c e m i a i n v i v o in c o m p a r i s o n w i t h n o r m a l r a t h e p a t o c y t e s k e p t a t 1.25 m m o l / L Ca 2+, P < .00005, or k e p t at 0.8 m m o l / L C a 2+, P < .00005. A s i g n i f i c a n t effect w a s f o u n d in r e l a t i o n to t h e dose of E G F , P < .002. I n n o r m a l r a t h e p a t o c y t e s , p e a k [ZH]thymidine i n c o r p o r a t i o n w a s a c h i e v e d a t 5 n g / m L E G F in 1.25 (P < .005), a n d 0.8 m m o l / L [Ca2+]e, (P < .005), respectively.

The data obtained during the current studies clearly show that, in hepatocytes obtained from normal rat livers, exposure to extracellular calcium concentrations comparable to those observed in hypocalcemia in vivo significantly perturbs the response to EGF as measured by both the [Ca2+]i increment and DNA synthesis responses. Long-standing hypocalcemia of D deficiency further exacerbates the defective response with a doseresponse curve to EGF for both the [Ca2÷]i increases and DNA synthesis activity well below that observed when extracellular calcium is acutely lowered in vitro. These observations confirm and extend the data obtained in previous studies from this laboratory where chronic hypocalcemia of vitamin D deficiency was shown to lower resting [Ca2+]i.s The current data clearly indicate that low extracellular calcium, but most particularly states of chronic hypocalcemia in vivo, decreases the hepatocellular capacity to respond to EGF. Our observations on the cellular response to EGF are also fully in line with an earlier study where hypocalcemic conditioning was demonstrated to lead to a significant decrease in hepatic EGF receptor density and autophosphorylation. 12 In addition, several in vivo and in vitro observations have already clearly shown that chronic hypocalcemia secondary to either D deftciency9 or thyroparathyroidectomy21-24 is associated with a defective hepatic regeneration phenomenon. Taken together, the results of these and the current

.•

2oo

C

•8-~5~ ~5o EGF significantly stimulated [~H]thymidine incorporation in normal rat hepatocytes in the presence of both normal and low [ C a 2 + ] e with peak [3H]thymidine incorporation occurring at the 5 ng/mL EGF dose at either 1.25 or 0.8 mmol/L extracellular Ca 2÷ (P < .005). However, decreasing [Ca2+]e from 1.25 to 0.8 mmol/L significantly reduced the DNA synthesis response to EGF (P < .004) in hepatocytes obtained from normal rat livers. Hepatocytes from hypocalcemic rats maintained in hypocalcemic conditions exhibited, however, a significantly lower [3H]thymidine incorporation response t h a n that obtained in hepatocytes from control rats cultured in normal (P < .00005), or low [ C a 2 + ] e ( P < .00005). Moreover, as illustrated in Fig. 7, the in vitro normalization of [Ca2+]e did not lead to the restoration of DNA synthesis in hepatocytes isolated from hypocalcemic rat livers. Hepatocyte viability was not affected by the culture conditions nor by hypocalcemic conditioning in vivo. Tyrphostin and verapamil, however, significantly decreased cell viability at the 48-hour point, preventing the studies aimed at investigating their effect on cell proliferation.

.~.~ ~J

100

r~

~ m

o 25

50

75

100

EGF (ng/ml) FIG. 7. Effect o f h y p o c a l c e m i c c o n d i t i o n i n g in v i v o on t h e in v i t r o [3H]thymidine i n c o r p o r a t i o n r e s p o n s e to E G F s t i m u l a t i o n in t h e p r e s e n c e of n o r m a l or low [Ca2÷]e. H e p a t o c y t e s were o b t a i n e d f r o m h y p o c a l c e m i c r a t s (n = 5) a n d m a i n t a i n e d in p r i m a r y c u l t u r e in p a i r e d conditions a t a n e x t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n of 1.25 ([~ - - [3), or 0.8 m m o l / L (ll - - 1 ) . C u l t u r e m e d i a were s u p p l e m e n t e d w i t h E G F a t c o n c e n t r a t i o n s r a n g i n g from 1 to 100 n g / m L , a n d all d e t e r m i n a t i o n s were m a d e in d u p l i c a t e d i s h e s . D a t a repres e n t t h e m e a n s _+ S E M for e a c h of t h e conditions u s e d . S t a t i s t i c a l l y s i g n i f i c a n t differences b e t w e e n g r o u p m e a n s were a n a l y z e d by a n a l y sis of co-variance, t a k i n g t h e r e s p o n s e in a b s e n c e of E G F as covariable. Effect of t h e e x t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n on [~H]t h y m i d i n e incorporation: NS.

1582

BILODEAU ET AL

studies thus strongly suggest that an altered EGF signaling system is most likely one of the major pathophysiological mechanisms associated with the suboptimal response to mitogenic signals associated with hypocalcemia. Our data also demonstrate that the decreased [Ca2÷]i and DNA synthesis responses found in hypocalcemia could not be overcome by increasing concentrations of EGF. This observation indicates that the factors liraiting responsiveness are not associated with EGF itself nor with defect(s) in the membrane sensitivity/affinity toward the growth factor. It supports the contention, however, t h a t the observed refractoriness to EGF is related, at least in part, to the density of its receptor, or to events distal to receptor binding such as autophosphorylation or protein tyrosine kinase activation, all of which have been shown to be decreased by hypocalcemia, le Collectively, these data thus indicate that hepatocytes isolated from hypocalcemic animals sustain a form ofhepatocellular resistance to the action of EGF. It is currently unknown, however, which of the components of the EGF-mediated calcium response (intracellular mobilization, or extracellular calcium entry) is mainly responsible for the low responsiveness to the growth factor when the [Ca2+]e is lowered in the hypocalcemic range. Indeed, the [Ca2÷]i response induced by growth factors such as EGF, TGF~, and HGF is thought to result from two converging systems: Ca 2+ release from intracellular pools 25'2~and Ca 2+ entry from the extracellular space through calcium channelsY Our data suggest that low extracellular calcium certainly contributed in decreasing the EGF-mediated [Ca2÷]i response as clearly demonstrated in the studies in which normal rat hepatocytes were exposed to the calcium channel blocker verapamil as well as the studies where normal hepatocytes were maintained in hypocalcemic conditions (0.8 mmol/L [Ca2÷]~), all of which resulted in a significantly inhibited rise in [Ca2+]~ without affecting the resting cytosolic Ca 2+ concentration. These observations were further confirmed using extracellular Mn 2+ as surrogate ion for calcium (as well as by 45Ca influx studies [data not shown]). Collectively, these data suggest that decreasing [Ca2÷]e or perturbing its cellular entry is sufficient to alter the EGFstimulated increase in [Ca2÷]~. However, the lower t h a n normal resting cytosolic Ca 2+ associated with chronic hypocalcemia could be an indication that the intracellular calcium pools might also be perturbed by longstanding calcium deprivation, both of which m a y well have compounded the effect of the low [Ca2+]e environment on the [Ca2+]~ and DNA synthesis responses, because, in several circumstances, hardly any elevation in [Ca2+]i and [3H]thymidine incorporation could be observed despite the application of supraoptimal concentrations of EGF. The already reported decrease in EGF receptor density and autophosphorylation 12 could well explain the refractoriness of hypocalcemic rat hepatocytes to the action of EGF as well as the failure of the in vitro attempt at normalizing [Ca2+]~ to reverse the

HEPATOLOGYJune 1995

in v i v o - m e d i a t e d effect of hypocalcemia on the EGFinduced mitogenic signal. Indeed, as already reported, the EGF receptor does not undergo upregnlation by de novo synthesis in vitro but rather is downregnlated in primary culture of hepatocytes, 2s an observation indicating that the in vivo effects of hypocalcemia, if linked to the EGF receptor, should not be expected to be overcome in vitro. Among the growth factors involved in hepatic compensatory hyperplasia, EGF/TGF, and HGF are now considered as the most powerful mediators responsible for initiating the events leading to cell proliferation. 3 However, calcium has also been clearly shown to be a necessary intracellular messenger for these hepatic mitogens. 2~32 Interestingly, a role of co-mitogens has been attributed to agents such as phenylephrine, vasopressin, and noradrenaline, which all have in common the property to mobilize [Ca2+]i in hepatocytes. 3-5 It is therefore tempting to postulate, in the light of the latter observations, t h a t these co-mitogens, by independently participating in the [Ca2+] i response, could very well potentiate the [Ca2+]i mobilization induced by EGF/TGF~ and HGF. Furthermore, these observations in conjunction with our data showing a defective [Ca2+]i response in hypocalcemia would also raise the hypothesis that the mitogenic potency of other growth factors, most particularly those inducing changes in [Ca2+]~y m a y also be perturbed by lower t h a n normal extracellular calcium concentrations. A significant inhibition of the EGF-stimulated increases in l e a 2 + ] i w a s also observed during the current studies in the presence of tyrphostin. Indeed, the increase in [Ca2+]i w a s almost completely abolished by tyrphostin at normal extracellular calcium concentration. These data show that autophosphorylation of the EGF receptor is, indeed, needed to fully support the EGF-mediated increase in [Ca2+]~ in normal circumstances, although tyrphostin m a y have compounded its inhibitory effect on EGF receptor autophosphorylation by also directly inhibiting Ca 2÷ entry as already evidenced in other systems. 34 The absence of a significant effect of tyrphostin and of verapamil at 0.8 mmol/L [Ca2+]o may be linked to the already reduced response to EGF that is observed at low extracellular calcium concentration. Mechanistically, it could be due to either a somewhat reduced autophosphorylation capacity of the EGF receptor that has already been shown to accompany hypocalcemic conditions 12 and/or to a requirement for calcium for complete inhibition of the EGF receptor autophosphorylation by tyrphostin. It m a y also indicate that, at low [Ca2+]e, EGF may have preferentially mediated a mobilization Ca 2÷ from intracellular stores that, in the current studies, are not expected to have been affected by the short in vitro conditioning period in 0.8 mmol/L [Ca2+]e. In fact, short-term exposure of normal rat hepatocytes to low [Ca2÷]e did not in any way influence the resting cytosolic Ca ~÷. The results of the current animal studies are relevant to several clinical situations. Indeed, both hypo-

HEPATOLOGYVol. 21, No. 6, 1995

calcemia and vitamin D depletion are common findings in patients with advanced liver diseases. In such conditions, however, the liver maintains its enzymatic capacity to respond to vitamin D administration by adequate production of 25-hydroxyvitamin D, 35'36 and the kidney is fully able to form the hormonal and active metabolite of the vitamin D endocrine system to allow normalization of the extracellular calcium homeostasis. Our data suggest that patients presenting nutritional or functional vitamin D depletion, if left untreated, may suffer an impaired hepatic regenerative capacity because of their associated marginal calcium status. Correction of hypocalcemia or vitamin D deftciency thus appears important, not only to counter the clinical manifestations of hypocalcemia, but also to preserve the capacity of the liver for compensatory growth in the light of its beneficial potential on the healing process. Our data thus indicate that extracellular calcium concentrations within the physiological concentration range can strongly influence the hepatocyte response to EGF. Indeed, a prevailing extracellular calcium concentration comparable to that of hypocalcemia in vivo is accompanied by a form of hepatocellular resistance to the cellular effect of EGF as illustrated by taking the [Ca2+]i response as marker of the early signal transduction, and DNA synthesis as outcome of the mitogenic signal.

Acknowledgment: The authors are grateful to JeanLuc Petit for his expert technical collaboration, to Andr~ Claude for the photography, and to Manon Cordeau for her excellent secretarial assistance.

BILODEAU ET AL

11.

12.

13.

14.

15.

16. 17. 18.

19.

20. 21.

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HEPATOLOGYJune 1995 33. Mine T, Kojima I, Ogata E, Nakamura T. Comparison of effects of HGF and EGF on cellular calcium in rat hepatocytes. Biochem Biophys Res Commun 1991; 181:1173-1180. 34. Lee KM, Toscas K, Villereal ML. Inhibition of bradykinin-induced and thapsigardin-induced (Ca2+) entry by tyrosine kinase inhibitors. J Biol Chem 1993; 14:9945-9948. 35. Plourde V, Gascon-Barr~ M, Willems B, Huet PM. Severe cholestasis leads to vitamin D depletion without perturbing its C-25 hydroxylation in the dog. HEPATOLOGY 1988;8:15771585. 36. Plourde V, Gascon-Barr~ M, Coulombe PA, Valli~res S, Huet PM. Hepatic handling of vitamin D3 in micronodular cirrhosis: a structure-function study in the rat. J Bone Miner Res 1988; 3: 461-471.