Stress causes alteration in attachment of rat hepatocytes to matrix protein substratum

146 Biochimica et Biophysica Acta, 1178 (1993) 146-152 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.0(I

BBAMCR 13432

Stress causes alteration in attachment of rat hepatocytes to matrix protein substratum

Susan Mathew and P.R. Sudhakaran Department of Biochemistry, Unit,ersity of Kerala, Kariavattom, Tril~andrum (India)

(Received 6 April 1993)

Key words: Matrix protein; Stress; Attachment kinetics; Heat shock; (Rat); (Hepatocyte)

The effect of different types of stress on the attachment of rat hepatocytes to various matrix protein substrata, such as collagen IV, fibronectin and laminin, was investigated. Study of the kinetics of attachment of adult hepatocytes subjected to heat-shock at 45°C for 30 rain showed significantly lower attachment to collagen IV, fibronectin and laminin substrata when compared to untreated controls. The alteration in attachment was observed after heat-treatment for 10 min and the extent of alteration appeared to increase with duration of the heat-treatment, as well as with increase in temperature. Foetal rat hepatocytes, which appeared to attach much more readily than adult rat hepatocytes to these substrata, particularly to laminin, also showed significantly lower attachment to all three substrata after heat-treatment at 45°C for 30 rain. This alteration in attachment appeared to be specific for matrix proteins as there was no significant effect on interaction of cells with non-matrix proteins, such as Con A and asialoglycoproteins. While the attachment of adult hepatocytes appeared to attain near control levels in about 4 h, the foetal cells recovered in about 2 h after heat-shock, indicating that the heat-shock effect is reversible and the recovery is faster in foetal hepatocytes. Other stress-causing agents, such as heavy metals, also caused an alteration in attachment of hepatocytes to these matrix proteins. Attachment of adult and foetal hepatocytes to collagen substratum was reduced by 100-/~M concentrations of heavy metals in the order Zn > Cd > La > As > Cu for adult cells and La > Zn > A s / C u > Cd for foetal cells. These heavy metals also caused significant reduction in attachment of adult hepatocytes to fibronectin and laminin, although the extent of inhibition was less than that for collagen substrata. However, these heavy metals did not significantly affect the attachment of foetal hepatocytes to laminin substratum indicating that the effect of heavy metals appeared to vary with the nature of the matrix protein substratum. On heat-shock, incorporation of [3H]leucine into cytoskeletal proteins such as cytokeratins CK8 and CK18 was reduced and significant amounts of two new proteins having an average molecular mass of 80 kDa and 90 kDa were found to be tightly associated with cytoskeletal proteins.

Introduction

The abil i ty of cells to a t t ach to the ex t race l lu la r mat r ix is cr i t ical to a n u m b e r of p h e n o m e n a , inc luding m a i n t e n a n c e of t issue integr i ty , wound heal ing, mor- phogen ic movemen t , ce l lu lar migra t ion and metas tas is . Ce l l - adhes ion sites have been iden t i f i ed u l t ras t ruc tu- ral ly as a reas of the cell surface th icken ing ad jacen t to the ex t race l lu la r mat r ix c o m p o n e n t s [1]. A large num- be r of molecu les have been iden t i f i ed as c o m p o n e n t s of these o rgan i sed s t ructures . They inc lude actin, c~- act inin, f ib ronec t in , tal in, v i n c u l i n / v i m e n t i n and o the r cy toske le ton -as soc ia t ed molecu les which are be l ieved to l ink act in f i l aments to the cell sur face [2-5], The ex t race l lu la r mo lecu le s in these a reas inc lude laminin , f ib ronec t in and h e p a r a n su lpha te p ro teog lycans . Aff in-

Correspondence to: P.R. Sudhakaran, Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum 695 581, India.

ity c h r o m a t o g r a p h y with ex t race l lu la r mat r ix adhesive g lycoprote ins , such as f ib ronec t in and laminin, use of an t ibod ie s to inhibi t cell adhes ion to matr ix glyco- p ro t e ins and d e m o n s t r a t i o n of the a d h e s i o n - p e r t u r b i n g an t ibod ie s in b locking b ind ing of mat r ix molecu les to m e m b r a n e p ro t e ins led to the ident i f ica t ion and local- izat ion of r ecep to r s for mat r ix molecu les on the cell sur face [6-8]. R e c e p t o r s for ex t race l lu la r matr ix molecu les be long ing to bo th in tegr in and non- in tegr in groups of p ro te ins have been ident i f ied in d i f ferent ce l lu lar systems [9].

Living organ isms are known to r e spond to a wide var ie ty of stress condi t ions which inc lude e leva ted tem- pe ra tu res , chemica ls and drugs such as alcohol , heavy meta ls , etc., in a var ie ty of ways [10,11]. A p a r t f rom the induct ion of specific s tress p ro t e ins [12], hea t - shock induces morpho log ica l changes in cul t iva ted cells and within a var ie ty of t issues, the severi ty of which is t e m p e r a t u r e - d e p e n d e n t [13]. The m e m b r a n e - a s s o c i - a t ed act in mic ro f i l amen t s in C H O cells were shown to

147

be heat-labile [14-17]. The structural integrity of the major elements of cytoskeleton, microtubule and intermediate filaments are also heat-labile [18]. The ability of CHO cells to reattach to individual matrix components such as vitronectin was shown to be reduced by heat and was correlated with the appearance of newly- synthesised proteins at the substrate-attachment site [13]. These changes in response to temperature appear to be cell and species-specific, as microfilament bundles in chick-embryo fibroblasts were not disorganised at temperatures at which CHO cell structures were affected [19].

Liver tissue is capable of overcoming the acute effects of injury by undergoing rapid regeneration. The interaction of hepatocytes with the components of the extracellular matrix is a critical event in the regeneration process. Further, since heat-shock reversibly dis- rupts microfilament bundles and alters the ability of cells to attach to substrata in a cell-specific manner, it was of interest to study how the attachment of hepatocytes to different matrix protein substrata was affected in response to stress.

Materials and Methods

All chemicals used were high-purity analytical-grade reagents. Minimum essential medium (Eagle's MEM), collagenase type IV, trypsin, Con A and p-nitrophenyl- N-acetyl-/3-D-glucosaminide were purchased from Sigma (St. Louis, MO, USA). Tissue-culture plas- ticwares were from M/s Nunc (Roskilde, Denmark). [3H]Leucine was a product of BARC (Bombay, India). Type-IV collagen and laminin prepared from EHS tumour were kindly provided by R.C. Hughes, NIMR (London, UK).

Cell-isolation techniques. Hepatocytes were isolated from adult rat liver (180-200 g, Sprague-Dawley strain) by collagenase perfusion by the method of Seglen [20] as described before [21]. Foetal cells were isolated after treating foetal livers with buffer containing 0.1% of each of collagenase and trypsin type III at 37°C for 10 min. Isolated cells were maintained in Eagle's medium supplemented with penicillin, streptomycin and insulin at 37°C in a humidified incubator (95% air/5% CO2). Viability was checked using 0.1% Try- pan blue in balanced salt solution (BSS) and only cell preparations with more than 95% viability were used.

Substrata. Cell attachment to various matrix and non-matrix proteins was studied. Fibronectin was prepared from plasma by affinity chromatography on gelatin-Sepharose [22] and asialoglycoprotein was prepared from bovine a2-macroglobulin by treatment with 0.05 M H 2 S O 4 at 80°C for 1 h [23]. Glass coverslips/multiwell plates were passively coated with 25 Fzg/ml of matrix proteins, such as collagen type-IV fibronectin or laminin, or with 200 Izg/ml of the non-

matrix proteins concanavalin A (con A) and asialoglycoprotein (ASG) for 3 h at room temperature prior to seeding cells. The free sites were blocked with 1% bovine serum albumin. The wells were washed with BSS thrice and finally once with MEM before plating the cells.

Treatment of cells. Cells were maintained at required temperatures for different time intervals in ther- mostated air incubators for heat-shock experiments. In heavy-metal stress experiments, the required concentrations of the metal salts in solution were added to the cells in medium and maintained at 37°C in a humidified air incubator (95% air/5% CO 2) for the required time intervals. The salts used were sodium arsenite, cadmium acetate, copper sulphate, lanthanum chloride and zinc chloride.

Cell-attachment assay. Attachment of hepatocytes to different substrata was measured by the method of Rubin et al. [24]. Hepatocytes (1 • 105 cells) suspended in MEM were seeded on coverslips/multiwells precoated with various proteins. The unattached cells were collected, washed once with PBS and both the attached and unattached cells were lysed in 0.1% Triton X-100 in 0.1 M citrate buffer (pH 4.6). The activity of /3- hexosaminidase in the cell lysate was assayed, using p-nitrophenyl-N-acetyl-/3-o-glucosaminide as substrate.

Metabolic labelling of cells. Hepatocytes were placed on collagen substrata and maintained at 37°C. The unattached cells were removed after 4 to 5 h and incubated in media containing hormones and antibi- otics overnight. The monolayer of hepatocytes was subjected to heat-shock at 45°C for 30 min in leucine- free medium and then labelled with 30 /zCi [3H]leucine/ml while maintaining the cells at 45°C for another 30 min. The cells were maintained in the same medium at 37°C for another 3½ h. The medium was removed and the washed monolayers of cells were lysed in buffer containing 0.5% Triton X-100, 2.5 mM EGTA, 0.6 M KC1, 14 mM /3-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride and 10 mM Hepes (pH 7.4). The insoluble proteins were washed in Tris-HCl (0.05 M, pH 6.8) and analysed by SDS-PAGE accord- ing to Laemmli [25]. Fluorography of the gel was per- formed using Indu X-Ray film [26]. The intensity of the individual protein bands were measured in a LKB laser densitometer. Radioactivity was measured in a LKB Rack Beta Liquid Scintillation Counter. Protein was estimated by the method of Lowry et al. [27].

Results

Effect of heat-shock on the attachment of adult hepatocytes to different substrata

In order to study whether the interaction of hepatocytes with the matrix protein substratum was affected in response to stress, hepatocytes isolated from adult

148

/ J " / - - ~ r *

AO ~ * • #

2O

0 - - -

9" > • 4 0 • j.~K

20

0 COlV

2O

0 iS ~,F- 90 T I M E ( I N MINUTES)

Fig. 1. Heat-shock alters the attachment of adult hepatocytes to different matrix proteins. Adult hepatocytes were heat-treated at 45°C for 30 min and plated on coverslips coated with 25 ~ g / m l of collagen IV (COIV), fibronectin (FN) and laminin (LN). Attachment to each substratum at 37°C at different time intervals was determined as described in Materials and Methods. The values given are the average of triplicate analysis and S.D. was less that 5%. (©)

untreated control and (o) heat-stressed.

rat liver were subjected to heat-shock and their attachment to coverslips coated with matrix proteins, such as fibronectin, laminin or type-IV collagen, was studied and the results are shown in Fig. 1. About 40% of the non-stressed cells added on to the collagen substrata, attached within 15 min and approx. 50% of the added cells attached after about 30-45 min. Studies on the kinetics of attachment of hepatocytes subjected to heat-shock at 45°C for 30 min showed significantly lower attachment to collagen substrata. Only about 20% of the stressed cells added to the substrata attached after 15 min. Like non-stressed cells, at 90 min after plating and at later time intervals, the extent of attachment to collagen substratum, remained at the maximum level. Like the collagen substratum, hepatocytes heat-stressed at 45°C for 30 min showed significantly lower attachment to fibronectin substratum when compared to the unheated control. Attachment of heat-treated hepatocytes to laminin coated coverslip was also significantly lower when compared to the control. Microscopic examination showed that the cells started to spread after about 60 min on collagen substratum and the extent of spreading increased with time. Heat-treated cells also started to develop spread/f lat morphology, but the rate of spreading ap-

t CO I~' FN LN ]

w m 80 I z u O .< <J

~- 404 i

0 c 10 20 30 C 10 20 30 10 20 30

DURATION OF STRESS (IN MINUTES)

Fig. 2. Increased duration of heat-stress resulted in corresponding decrease in attachment. Adult hepatocytes were heat-treated at 45°C for 10, 20 and 30 rain and plated on coverslips coated with 25 # g / e l of collagen IV (COIV) fibronectin (FN) and laminin (LN). Attach- ment to each substratum at 37°C after 30 rain was determined, The

values given are the average of triplicate analysis.

peared to be slow when compared to control. The cells attached on to fibronectin and laminin appeared to show a flat morphology after about 120 rain and the extent of spreading of heat-treated cells on these substrata was less during this time interval. After about 5 - 6 h both heat-stressed and control cells appeared to be maximally spread on all substrata.

The duration of stress was varied by exposing cells to 45°C for different time intervals and the attachment of these cells to collagen, fibronectin and laminin substrata was also studied. With increase in the duration of stress, there was corresponding decrease in the number of cells attached 30 min after plating to each substrate (Fig. 2). Even 10 min heat-treatment at 45°C produced a decrease in attachment to each substrate. This was further examined by subjecting the cells to heat-treatment for different temperatures. Heat treatment for 30 min at 42°C produced 10% decrease in attachment to collagen but the attachment to fibronectin and laminin, though reduced, was not significant. But on further increase in temperature to 45°C or 52°C there was a considerable reduction in the extent of cell attachment to these substrata after 45 min (Fig.

uJ~ 80' 3 " z ~-© ~.D(J < 40 ~

0

CO !V FN LN

i

C 42 45 52 C 42 45 52 C 42 45 52 TEMPERATURE (°C)

Fig. 3. Cellular attachment decreased with increase in intensiy of heat-stress. Adult hepatocytes were heated at 42°C, 45°C and 52°C for 60 rain and were plated on coverslips coated with 25 # g / m l collagen (COIV), fibronectin (FN) and laminin (LN). The attachment to each substratum at 37°C after 30 min was determined and expressed as percentage of untreated control. The values plotted are

the mean of triplicate analysis. S.D. < 5~,.

149

3). These results indicated that the alteration in attachment of hepatocytes to different matrix substrata in response to heat-shock depends on the degree of stress imposed on the cells.

Effect of heat-shock on the attachment of foetal hepatocytes

Foetal rat hepatocytes appeared to attach much more readily than adult hepatocytes. Maximum attachment was observed 30-45 min after seeding of cells. Of the different substrata, foetal hepatocytes appeared to attach more to laminin after 45 min. Heat treatment of cells at 45°C for 30 min significantly reduced the attachment of cells to all the three substrata (Fig. 4).

Effect of heat-stress on the attachment of hepatocytes to non-matrix protein substrata

In order to examine whether the alteration in attachment observed on heat-shock was a specific process related to the interaction of cells with the matrix proteins, attachment of hepatocytes after heat-stress to non-matrix proteins, such as Con A and ASG, was studied. Con A can bind with cell-surface glycoproteins

z 20 b,J

T L,)

0 --

o

2O

0 • - -

30 60 90

T IME (IN MINUTES)

Fig. 5. Attachment of hepatocytes to concanavalin A (Con A) and asialoglycoprotein (ASG)-coated substrata was unaltered by heat- shock. Adult hepatocytes were stressed at 45°C for 30 rain and plated on coverslips coated with 200/~g/ml Con A or ASG. The percent of cells attached for various time intervals at 37°C was determined. The values given are the average of triplicate analysis and S.D. was less

than 5%. (o) control and (o) stressed.

60-

40.

20

0 I -

z 60. h i

2r " r

40- i - -

20-

0 '

60.

40-

20~

LN

C0 IV

15 3-0 45 TIME (IN MINUTES)

Fig. 4. Foetal hepatocytes show a decrease in attachment to different matrix proteins after heat-shock. Foetal hepatocytes were heat- stressed at 45°C for 30 min and plated on coverslips coated with 25 /zg/ml collagen IV (COIV), fibronectin (FN) and laminin (LN). The percent of cells attached at 37°C for various time intervals was determined. The values plotted are the mean of triplicate analysis.

S.D. < 5%. (o) untreated control; (e) heat-stressed.

[28] while ASG binds with great affinity to the ASG receptor on hepatocytes [29]. Coverslips coated with Con A and ASG were used as substrata for the attachment of hepatocytes. There was no significant difference in the kinetics of attachment between non-stressed and heat-treated cells onto these substrata (Fig. 5), indicating that the alteration in attachment is probably specific for matrix proteins. Hepatocytes, attached to these substrata, showed a normal round morphology and did not spread, even after about 8 h, probably indicating that in both control and stressed cells cytoskeletal structures have not been formed under these conditions.

Recovery from heat-stress The reversibility of heat-shock effect on the attach-

ment of hepatocytes to matrix proteins was examined by studying the attachment to collagen at different time intervals after heat-shock. Adult hepatocytes appeared to recover slowly after heat-shock (Fig. 6). At 3½ h after heat-treatment (3 h recovery at 37°C plus 30 min attachment at 37°C) the attachment to collagen was about 80-85% of the untreated control. But the foetal hepatocytes appeared to recover faster than the adult hepatocqctes. Attachment of foetal hepatocytes was similar to the control after about 2 h, indicating complete recovery after heat-shock. A higher percentage of foetal ceils attach to all matrix-protein substrata than do adult hepatocytes for each time period in the recovery phase, indicating that foetal cells recover faster than the adult ceils after heat-shock.

150

3 0 r r ~I00 0 U

0

~ 50 Z UA

I

<

0

J

J J

1 2

TIME (tN HOURS)

Fig. 6. Foetal hepatocytes recovered faster than adult cells from heat-shock. Adult ( • ) and foetal (o) hepatocytes were stressed for 1 h at 45°C and allowed to recover at 37°C for different time intervals. 1.105 cells were seeded on coverslips coated with 25 txg/ml collagen IV and allowed to attach at 37°C. The attachment of cells for 30 rain was expressed as percentage of the untreated controls maintained under identical conditions at 37°C for the respective time intervals. The values given are the mean of triplicate analysis. S.D. was less

than 5%.

Effect of heavy-metal stress on attachment of hepatocytes to matrix proteins

As cells were reported to be stressed on exposure to heavy metals, as does heat-shock as manifested by the production of identical type of stress proteins [30], the effect of metals such as cadmium, arsenic, lanthanum, zinc and copper on the a t tachment of foetal and adult

B O ~ TM 40

c r

12)

u_u ~ FN o g0 g

~ 4o tlJ

I u

< 0 t---

< 80 1 i - - I ico lv

0 As Cd Cu Lo Zn

Fig. 7. Some heavy metals decrease the attachment of foetal and adult hepatocytes to matrix proteins. Foetal ([]) and adult (D) hepatocytes were plated on coverslips coated with 25 txg/ml of collagen IV (COIV), fibronectin (FN) and laminin (LN). 100 IxM concentration each of As, Cd, Cu, La and Zn were added to the medium containing cells and maintained for 60 min. Attachment at 37°C was determined. The untreated control cells attached to substratum was taken as 100%. The values given are the average of

triplicate analysis. S.D. < 5%.

hepatocytes to different matrix proteins was studied, and the results are given in Fig. 7. At tachment of foetal and adult hepatocytes, 60 min after plating on collagen substratum was significantly reduced by 100 # M concentration of cadmium, arsenic, zinc, lanthanum and copper ions. The maximum effect was observed for Zn, Cd and La and minimum for Cu in the case of adult hepatocytes. La caused maximum reduction of attachment of foetal hepatocytes and Cd the least. In the case of both adult and foetal hepatocytes, t reatment with these heavy metals caused significant reduction in at tachment of cells to fibronectin substratum also, ex- cept in the case of copper for adult hepatocytes. The inhibitory effect of these heavy metals on at tachment of cells to fibronectin appeared to be less than that for collagen substrata. However, these heavy metals did not significantly affect the a t tachment of foetal hepatocytes to laminin substrata. Attachment of adult rat hepatocytes to laminin substratum was significantly

C S

200

116

97

ck 8 ck 18

6 7

4 5

Fig. 8. Adult hepatocytes were plated on multiwells precoated with 25 #g /ml collagen IV and maintained at 37°C. Untreated cells were removed after 4 h and the cells were maintained in culture at 37°C overnight. The monolayer of cells was preincubated in leucine free MEM and heat-treated at 45°C for 1 h and labelled with 30/zCi/ml of [3H]leucine for 4 h at 37°C and the insoluble cytoskeletal proteins were isolated as described in the text. The samples were analysed using 10% SDS-PAGE. The gels were stained and prepared for fluorography, (C), Control; (S), stressed; (CK), Cytokeratins 8 and 18:

(-), new bands.

151

reduced on treatment with As, Cd, and to a lesser extent La, but not by Cu or Zn. These results indicated that the heavy metals affect the attachment of foetal and adult hepatocytes to matrix proteins, but the effect appears to be different for different heavy metals and it varied with the nature of the matrix-protein substratum.

Synthesis of cytoskeleton-associated proteins on heat- shock

The formation of the cytoskeletal elements in heat- stress was studied biochemically. Monolayers of hepatocytes maintained in culture were heat-shocked at 45°C and the cells were labelled with [3H]leucine. The insoluble cytoskeletal proteins were collected after treatment with detergent and salt. Incorporation of [3H]leucine into total cytoskeletal protein, 4 h after heat-shock, was about 45% of that of the control. On electrophoresis, followed by fluorography, apart from a lower amount of radiolabelled cytoskeletal proteins, such as actin and cytokeratins CK8 and CK18 in heat- shocked cells, two new protein bands corresponding to average molecular masses of 80 kDa and 90 kDa were also seen (Fig. 8). These proteins appeared to be present in trace amount in control-cell preparations also. The fact that these proteins are resistant to solu- bilization by salt and detergent along with a disul- phide-reducing agent and cation .chelator suggested that significant amounts of these proteins are tightly held with other cytoskeletal components. This indicated a possible role for these proteins in the organisation of the cytoskeletal components.

Discussion

The results described above indicate that heat-shock affects the ability of hepatocytes to attach to matrix protein substrata. This effect appears to be reversible and depends on the severity and duration of stress. These results are similar to the alteration in attachment of CHO cells to vitronectin in response to heat- treatment at 45°C [13] and that of fibroblasts to tissue- culture surface [31]. The altered attachment to matrix proteins observed here does not appear to be a direct effect of heat on hepatocyte surface, as (1) attachment to non-matrix proteins such as Con A and ASG was unaffected by heat-shock; (2) heavy metals such as cadmium, lanthanum, zinc, arsenic, etc., which are known to cause stress, as do elevated temperatures, also cause an alteration in the hepatocyte attachment to matrix proteins. Further, iodination of the hepatocyte surface of heat-shocked and untreated cells followed by electrophoresis and fluorography did not show any significant difference in the cell-surface protein profile (data not shown). But it is not clear whether the heavy metals cause alteration in attachment by the

same or different mechanisms as heat-stress. The observation that attachment to non-matrix proteins was not affected by heat-shock suggests that probably heat-shock alters attachment of hepatocytes to matrix proteins by affecting the cytoskeletal and microtubular elements and the organisation of adhesion sites. Re- covery experiments indicated that the changes are reversible. Fluorescence microscopy studies had shown that actin microfilament bundles of CHO cells are heat-labile and following heat-shock, the prominent microfilament bundles which spanned the cytoplasm almost disappeared [14-17]. Cress et al. [13] observed that the appearance of two proteins, p150 and p82, in the substratum-attached material was delayed by heat- shock and suggested that these proteins may be involved in the attachment of CHO cells to vitronectin and other substrata. But we did not get any significant amounts of proteins in this range of molecular mass in the substrate-attached material of the heat-shocked hepatocytes plated on fibronectin, laminin or collagen IV. The heat-induced alteration in adhesion of hepatocytes to matrix proteins may be due to a delay in the processing of receptors for these matrix proteins to the membrane, consequent upon the disorganization of cytoskeletal and microfilament bundles. The observation that foetal hepatocytes recover faster than adult hepatocytes may indicate that the reorganization of these cytoskeletal and microfilament bundles and the processing of the receptors take place faster in the foetal cells. A faster rate of processing and /or increased number of membrane receptors for matrix proteins in foetal hepatocytes can also result in a faster recovery after heat-shock. The observation that foetal cells attached more to laminin is similar to the earlier report where comparison of kinetics of attachment of hepatocytes from regenerating rat liver to laminin and fibronectin was made and suggested the possibility of a higher number of receptors for laminin on hepatocytes in regenerating liver [32].

A variety of cells have been shown to produce stress proteins belonging to HSP60, HSP70 and HSP90 family in response to heat-shock and heavy metals such as arsenic [33]. Stress proteins belonging to HSP100 family have been shown to bind to actin filaments [34]. It is not known whether these proteins are involved in the reassembly of cytoskeletal elements during the recovery phase. It has been found that in heat-shocked hepatocytes two proteins having molecular masses of around 80 kDa and 90 kDa, presumably belonging to the heat-shock protein groups were tightly-bound to intermediate-filament-rich cytoskeleton. The presence of trace amounts of these proteins in non-stressed cells in association with cytoskeleton suggests that probably they are involved in the organisation of the cytoskeletal elements during the recovery phase. This aspect is being studied in detail. Hepatocytes recovering after

152

heat-shock may be a useful system to study the organisation and assembly of cytoskeleton and the possible involvement of heat-shock proteins in this process.

Further, our own data (Mathew, S. and Sudhakaran, P.R., unpublished data), as well as others, showed that in response to heat-shock and heavy metals there is a rapid intracellular flux of free calcium ions [35,36]. Calcium ions through calmodulin are reported to be involved in the disassembly of microtubules [37] and in the disassociation of actin filaments [38,39]. These events may cause an alteration in the interaction of the cells with the matrix protein during the early stages of heat-shock. A reduction in the free calcium on removal of stress may allow the assembly of these structures and promote the attachment in the recovery phase.

But all the heavy metals tested did not produce identical effects on attachment of hepatocytes to different matrix proteins, particularly in the case of foetal hepatocytes to laminin. In hepatocytes, multiple receptors for laminin belonging to both the non-integrin family [40], and the integrin family [41] are reported to be present. Integrin receptors for different matrix proteins have been shown to have a varying divalent-cation-dependence [42]. A 67 kDa protein which binds with laminin in a cation-independent manner was found to be present in foetal liver (Kumar, A. and Sud- hakaran, P.R., personal communication). The possibility that the affinity of these receptors to laminin are differentially affected in response to heavy metal stress cannot be excluded.

Acknowledgements

Financial assistance received from DST, Govern- ment of India, New Delhi is gratefully acknowledged. S.M. is a Junior Research Fellow of University Grants Commission, New Delhi.

References

1 Abercrombie, M., Heaysman, E.M. and Pegrum, S.M. (1971) Exp. Cell Res. 67, 359-367.

2 Wehland, J., Osborn, M. and Weber, K. (1979) J. Cell Sci. 37, 257-273.

3 Burridge, K. and Feramisco, J. (1980) Cell 19, 587-595. 4 Geiger, B., Tokuyasu, K.T., Dutton, A.H. and Singer, S.J. (1980)

Proc. Natl. Acad. Sci. USA 77, 4127-4131. 5 Burridge, K. and Connell, L. (19831 J. Cell Biol. 94, 359-367. 6 Wylie, D.E., Damsky, C.H. and Buck, C.A. (1979) J. Cell Biol. 80,

385-401. 7 Kohler, G. and Milstein, C. (1975) Nature 256, 495-497. 8 Malinoff, H.L. and Wicha, M.S. (1983) J. Cell Biol. 96, 1475-1479.

9 Buck, C.A. and Horwitz, A.F. (1987) Annu. Rev. Cell Biol. 3, 179-205.

10 Southgate, R., Mirault, M.-E., Ayme, A. and Tissieres, A. (1985) in Changes in Eukaryotic Gene Expression in Response to Envi- ronmental Stress (Pitkinson, B.G. and Walden, D.B., eds.), pp. 3-5, Academic Press, New York.

11 Lindquist, S. (1986)Annu. Rev. Biochem. 55, 1151-1191. 12 Welch, W.J. and Suhan, J.P (1986) J. Cell Biol. 1(13, 2035-21152. 13 Cress, A.E., Majda, J.A., Glass, J.R., Stringer, D.E. and Gerner,

E.W. (1990) Exp. Cell Res. 190, 40-46. 14 Welch, W.J. and Feramisco, J.R. (1985) Mol. Cell Biol. 5, 1571-

1581. 15 Glass, J.R., Dewitt, R.G. and Cress, A.E. (1985) Cancer Res. 45,

258-262. 16 Welch, W.J. and Suhan, J.P. (1985) J. Cell Biol. 101, 1198-1211. 17 Van Bergen en Henegouwen, P.M.P. and Linnemans, W.A.M.

(1987) Exp. Cell Res. 171,367-375. 18 Shyy, B., Asch, B.B. and Asch, H.L. (1989) J. Cell Biol. 1(18,

997-1008. 19 Collier, N.C. and Schlesinger, M.J. (1986) J. Cell Biol. 1(/3,

1495-1507. 20 Seglen, P.O. (19761 Methods Cell Biol. 13, 29-78. 21 Sudhakaran, P.R., Sinn, W. and Von Figura, K. (1980) Biochem.

J. 192, 395-402. 22 Engvall, E. and Ruoslahti, E. (1977) Int. J. Cancer. 20, 1-5. 23 Spiro, G.R. (1966) Methods Enzymol. 8, 3 25. 24 Rubin, K., Oldberg, A., Hook, M. and Obrink, B. (19781 Exp.

Cell Res. 117, 165-177. 25 Laemmli, U.K. (1970) Nature 227, 680-685. 26 Chamberlain, J.B. (1979) Anal. Biochem. 98, 132-135. 27 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.

(19511 J. Biol. Chem. 103, 265-275. 28 Agrawal, B.B.L. and Goldstein, I.J. (19721 Methods Enzymol. 28,

313. 29 Ashwell, G. and Harford, J. (1982) Annu. Rev. Biochem. 51,

531-554. 30 Levinson, W., Oppermann, H. and Jackson, J. (1980) Biochim.

Biophys. Acta 606, 170-180. 31 Lin, P.S., Butterfield, C.E. and Wallach, D.F. (1977) Cell Biol.

Int. Rep. 1, 57-61. 32 Carlsson, R., Engvall, E., Freeman, A. and Ruoslahti, E. (1981)

Proc. Natl. Acad. Sci. USA 78, 2403-2406. 33 Johnson, D., Oppermann, H., Jackson, J. and Levinson, W.

(19801 J. Biol. Chem. 255, 6975-6980. 34 Koyasu, A., Nishida, E., Miyata, Y., Sakai, H. and Yahara, 1.

(19891 J. Biol Chem. 264, 15083-15087. 35 Mosser, D.D., Kotzbauer, P.T., Sarge, K.D. and Morimoto, R.I.

(1990) Proc. Natl. Acad. Sci. USA 87, 3748-3752. 36 Welch, W.J., Garrels, J.I., Thomas, G.P., Lin, J.J-C. and

Feramisco, J.R. (1983) J. Biol. Chem. 258, 7102-7111. 37 Nishida, E., Kumagai, H., Ohtsuki, I. and Sakai, H. (19791 J.

Biochem. 85, 1257-1266. 38 Duhaiman, A.S. and Bamburg, J.R. (19841 Biochemistry 23,

1600-1608. 39 Rosenberg, S., Stracher, A. and Burridge, K. (1981) J. Biol.

Chem. 256, 2986-2991. 40 Clement, B., Segui-Real, B., Savagner, P., Kleinmann, H.K. and

Yamada, Y. (1990) J. Cell Biol. 110, 185-192. 41 Forsberg, E., Paulsson, M., Timpl, R. and Johansson, S. (1990) J.

Biol. Chem. 265, 6376-6381. 42 Elices, M.J., Urry, L.A. and Hemler, M.E. (1991) J. Cell Biol.

112, 169-181.

Documents

Stress causes alteration in attachment of rat hepatocytes to matrix protein substratum