Primed in Sweden Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved ISSN 0014-4827
Experimental Cell Research 110 (1977) 419425
CELL
ATTACHMENT
TO COLLAGEN:
ROBERT J. KLEBE, JAMES R. HALL, Division
PAMELLA
THE IONIC
REQUIREMENTS
ROSENBERGER and W. DARYL DICKEY
of Human Genetics, Department of Human Biological Chemistry and Genetics, University of Texas Medical Blanch, Galveston, TX 77550, USA
SUMMARY This study is concerned with three aspects of the ionic requirements for cell attachment to collagen; namely (a) the divalent, (b) monovalent cation specificities and (c) pH optima for cell interaction with collagen. The divalent cation requirement for cell attachment to collagen can be sufliced by Ca*+, MgP+ and certain transition group metals; whereas Baz+, SrP+, and polyamines are inactive. The pH optimum for cell attachment in this system occurs in the physiological range. The monovalent cation requirement for cell attachment to collagen is satisfied by isotonic NaCl, KCl, LiCl, NH,Cl, sucrose, and glucose. Pronounced inhibition of cell attachment occurs under both hypertonic and hypotonic conditions.
Over the last few years, several laboratories cation requirement for cells to attach to the have demonstrated proteins that mediate c-CAP-collagen complex and (3) requirecell adhesion [l-lo]. This study is con- ment for cellular metabolic energy [6,7]. cerned with the ionic requirements of a The present investigation is concerned serum-derived collagen-dependent cell at- with three aspects of the ionic requirements tachment protein (c-CAP). Several aspects for the interaction of cells with a c-CAPof the mechanism of action of c-CAP have collagen complex, namely (a) divalent; (b) been previously described. Cell attachment monovalent specificities; and (c) pH optima to collagen requires (a) a high molecular for cell attachment. weight serum protein, (b) divalent cations, (c) an energy source (glucose) and (d) bufMETHODS AND MATERIALS fered saline [6,7]. The cell attachment pro- Maintenance of cell lines tein binds preferentially to the CB, cyano- In the experiments described below, CHO Chinese gen bromide peptide of the cY,-chain of type hamster ovary cells [18] and a mutant of CHO, termed CHOatt- [9], were employed. CHOatr- is defective in I collagen [8]. c-CAP binds to collagen in its adhesive response to purified c-CAP as well as to the absence of divalent cations; in contrast, both Ca*+ and MgP+ [9]. Due to the fact that CHO is a occurring L-proline auxotroph, growth medivalent cations are required for cultured naturally dium must either contain or be supplemented with cells to attach to a c-CAP-collagen complex L-proline [18]. Both the wild type and mutant CHO lines were routinely maintained with Dulbecco’s [6]. Hence, three steps in cell attachment to cell modified Eagle’s medium supplemented with 1 mM collagen can presently be identified; namely L-proline, 10% fetal calf serum (FCS), and 100 U/ml plus 100 &ml streptomycin. Cells were (1) binding of c-CAP to collagen (no di- penicillin maintained at 36S”c under a 10% CO,-in-air atmovalent cation requirement), (2) divalent sphere. Exp
Cell
Res
I10
(1977)
420
60
Klebe et al.
I
04 0
,
, 2
, 4
,
, 6
, 6
, 10
pH; ordinate: % cell attachment. pH optima for the process of cell attachment to collagen. The effect of pH on (a) binding of cell attachment protein (c-CAP) to collagen (X-X) and (b) cell attachment to a preformed c-CAP collagen complex (0-O) were studied as described in the text. The buffer ions which were employed at 10 mM were glycine OpH 1.85, pH 2.3, and pH 9.8), sodium acetate @H 4.8), MES @H 6.15), imidazole (PH 6.95), potassium phosphate (pH 7.2), Hepes @H 7.5), Bicine (PH 8.35), and borate (pH 9.2). As indicated above, c-CAP binds to collagen over a broad pH range while cell attachment to a pre-formed c-CAP-collagen complex takes place optimally in the physiological range.
Fig.
1. Abscissa:
Minimal attachment medium (MAM) A minimal attachment medium, termed MAM medium [6], has been employed in. the experiments described below. MAM medium contains 116 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 1 mM CaCl,, 1 mM MaCl,. and 10 mM Henes, DH 7.5. In exneriments co&erned with the requirements for divalent cations, Ca*+ and Mgl+ were either deleted or varied in most experiments. In studies concerned with the pH optimum for cell attachment, other buffer anions were employed, within 0.5 pH units of their respective pK’s, in place of Hepes. The buffer ions were tested at pH 7.5 in order to rule out any inhibitory effect of the buffer ion itself in pH optimal experiments. The buffers. which were emnloved (at 10 mM). are aiven in fg ‘1. In studies co&&ning the monovalent cation reauirement for cell attachment. the NaCl and KC1 components of MAM medium were either deleted or varied as described in the text.
Cell attachment to collagen assay The assay for cell attachment to collagen has been described in detail [6]. In brief, the assay procedure is as follows. c-CAP was permitted to bind to rat-tail collagen-coated bacteriological Petri plates in MAM medium. c-CAP purified to the 2nd PEG step [6] was employed. After an incubation period that permits c-CAP to bind to collagen, cultured cells were added to the c-CAP treated Petri plates and incubated at Exp Cell Res II0 (1977)
37°C for 1.5 h. At the end of the assay period, those cells that had attached were trypsinized and counted with an electronic cell counter. In the absence of c-CAP or an active divalent cation, no cell attachment to collagen is observed; in the presence of c-CAP and an active divalent cation, the number of cells attached is a function of the c-CAP or active divalent cation concentration [6]. In the studies described, the following technical modifications of the previously reported assay procedure [6] were employed. (1) Plates were not incubated in a CO, environment since the heavv metal ions studied are often precipitated as carbonates when CO, is oresent. (2) Since several oroteins 111-131and mammalian cells cl4, 151undergo-physical-adsorption to glass and plastic surfaces when exposure is carried out under protein-free conditions, Petri plates were pretreated with protein in order to avoid artifacts arising from physical adsorption. Thus, in experiments that involved deletion of protein from media, collagenized Petri plates were treated with 100 pg/ml bovine serum albumin (BSA) and washed three times with MAM medium (minus divalent cations). The BSA treatment renders the collagenized Petri plates wettable and minimizes the adsorption of other proteins and cells. (3) Cells were washed orior to the beginning of the assay with either (a) MA-M medium minus dc valent cations (in the case of experiments involving divalent cations); or (b) with MAM medium containing a single monovalent cation or carbohydrate at isotonic concentration (in the case of experiments involving monovalent cations).
Reagents All monovalent and divalent cations employed were in the chloride form. with the excention of Bex+ which was obtained as the sulfate. All chemicals employed were of reagent grade.
RESULTS pH Optimum for (a) cell attachment protein binding and(b) cell attachment to a c-CAP-collagen complex The pII optimum for cell attachment to collagen was determined by assessing the effect of pH on (a) the binding of c-CAP itself to collagen; (b) the attachment of cells to a pre-formed c-CAP-collagen complex. c-CAP is stable for at least 1 h in a range from pH 3 to 10.5. In order to determine the infhrence of pH on the binding of c-CAP to collagen, a saturating amount of purified c-CAP, equivalent in activity to 5 % calf serum, was ex-
421
Ionic requirements
Petri plates were (a) treated with a saturating amount of purified c-CAP at pH 7.5 in complete MAM medium: (b) washed twice with saline; (c) cells were then allowed to attach in modified MAM medium at PI-I’S established with the buffer ions given in the fig. 1. The pH optimum for cell attachment to a c-CAP-collagen complex was found to be narrower than the pH range in which c-CAP itself binds to collagen (fig. 1). 0
Fig.
001
063
2. Abscissa:
0’1
0’3
molar cont.;
ordinate:
%
cells at-
tached. Cell attachment to collagen was studied in the presence of several salts of monovalent cations or carbohydrates at the concentrations indicated above. The compounds tested for their ability to promote cell attachment were NaCl (0-O); KC1 (A-A); NH&l (O-O); LiCl (X-X); alucose (0-O): and sucrose (6-d). Above the &o&tic concentration, cells became crenated. Below the isotonic concentration, cells were microscooicallv observed to become increasinaly swollen as ihe salt or carbohydrate concentrati& was decreased. It should be noted that cell attachment was markedly inhibited at hypotonic concentrations that did not result in noticeable cell lysis. As indicated above, cell attachment to collagen is optimal at the isotonic concentration (about 0.15 M for salts and 0.3 M for carbohydrates).‘ Increasing inhibition of cell attachment occurs as the medium becomes more hypotonic or hypertonic.
posed to collagen-coated Petri plates at PI-I’S established with the buffer ions given in fig. 1. After a 1 h incubation at 37°C the c-CAP treated plates were (a) washed twice with saline to remove excess c-CAP, (b) washed with complete MAM medium (pH 7.5) and- (c) cell attachment was then assayed as described in the Methods. Under the conditions described above, c-CAP was found to bind to collagen between pH 1.95 to pH 9.8; however, maximum c-CAP binding occurred in the physiological range (fig. 1). In order to determine the pH range in which cells will bind to a pre-formed c-CAP-collagen complex, collagen-coated
Monovalent cation requirement for cell attachment to collagen
The monovalent cation requirement for cell attachment to a c-CAP-collagen complex wag studied
by (a)
treating
cofi~en-coated
Petri plates with a saturating level of purified c-CAP in complete MAM medium, (b) washing twice with MAM medium lacking monovalent cations and (c) adding CHO cells to c-CAP treated plates containing MAM
medium
modified
to include
only in-
dividual monovalent cations or carbohydrateg,
When employed
at or near their iso-
tonic concentrations, several monovalent cations and carbohydrates were capable of supporting cell attachment (fig. 2). Divalent cation requirement for cell attachment to collagen
We have previously shown that Ca2+ and/ or Mg2+ are required for cell attachment to collagen [6]. Several other di- and trivalent cations were tested for their ability to promote cell attachment to collagen. Those multivalent cations that formed precipitates at or below 1 mM were excluded from the study. The cations studied are presented in an order of increasing ionic radius in table 1 and have been divided into three classes on the basis of their cell attachment properties. Exp Cell Res 110 (1977)
422
Klebe et al.
Table 1. Di- and trivalent cation requirement for 50 % cell attachment to collagen Divalent cation”
Crystal ionic radius b
ClassI BePfd AP+ d Fe3+d
0.35 A
5.7
0.51 0.64
5.8 4.6
Class ZZ MiP+ Ni2+ co*+ Zd+ Mn2+ Ca2+
0.66A 0.69 0.72 0.74 0.80 0.99
3.0 2.9
Class III se+ Ba2+
CHOa”-
CHO Z/R Ratioc
+ c-CAP
3x10-*
:.:: 2:5 2.0
1.12A
1.8
1.34
1.5
-c-CAP
+c-CAP
-c-CAP
6x lo+ mM
3x 1OV mM 10-Z ax 10-S
3x lo-* mM
8X 10-Z
>lOmM > 10e >lO > 10e >lO >lO
3mM >lO’ 1 >1oe 3x 10-Z 100
>lOmM > 10” >lO > 1oe >lO >lO
>lO mM >lO
>lOmM >lO
>lOmM >lO
>lO mM >lO
mM
10-Z 10-Z
10-Z 10-Z
lo-* mM 2x10-1 6x lo+
10-l
8x lo+
10-Z 10-Z
Cell attachment assays were carried out as described in the text. Collagen coated Petri plates were treated with 100 &ml BSA for 30 min and washed twice with MAM (minus divalent cations) prior to use in the assay. Minimal Attachment Medium (MAM) minus divalent cations was employed for washing cells. In the case of those divalent cations that promoted cell attachment to collagen, the concentration required for 50 % cell attachment is entered. a Multivalent cations which formed precipitates at or below 1 mM were excluded from the study. b Crystal ionic radii were taken from [ 191. c Z/R is the charge to radius ratio. d Cells cannot be removed by trypsin. e Precipitate formed at 10 mM.
Class I. The di- and trivalent ions in class I are smaller in ionic radius than MgZ+ and were found to attach cells in the presence or absence of c-CAP (table 1). In addition, ions of class I avidly promoted the attachment of a mutant (CHOati-) that is defective in cell attachment to collagen [9] (table 1). Cells attached to collagen by ions in class I were found to be resistant to dislodgement from collagen by both trypsin and EDTA. Cells attached to collagen by 0.1 mM Be2+ were found to resist removal from collagen for at least 16 h by 1000 times the trypsin concentration required to remove Ca2+-attached cells in 15 min. Similar results were obtained in the case of A13+and Fe3+. Class ZZ. The divalent cations in class II range in ionic radius between that of Mg2+ and Ca2+. Divalent cations of class II reExp CellResIIO (1977)
quired c-CAP in order to attach cells to collagen (table 1). As is indicated in table 1, CHOatt-, which is genetically altered in its response to Ca2+and Mg2+ [9], was also defective in its response to Ni2+, Co2+, Zn2+, and Mn2+. Class ZZZ. Divalent cations in class III are larger in ionic radius than Ca2+. These ions did not promote cell attachment to collagen (table 1). Polyamines. Since the divalent metal ion requirement of certain enzymes can be sufficed by polyamines [16, 171, several multivalent amino compounds were investigated as replacements for metal ions in the cell attachment process. At concentrations as high as 10 mM, hydrazine, ethylenediamine, putrescine, and spermidine were not able to substitute for divalent metal ions.
Ionic requirements
423
Table 2. Divalent cation activation and inhibition of enzymes Enzyme
Activators
Carboxypeptidase A (a) Esterase activity
Inhibitors
(6) Peptidase activity
Cde+>Hg*+>Zn2+>Co2+ >Ni2+>MnZ+ CoZ+>Ni2+>ZnZ+>Mn*+
Pyruvate kinase (a) Pyruvate reaction (6) Hydroxylamine reaction
Mg*+, Mn*+ Co2+>ZnZ+>Mnz+
Carbonic
Ref.
1351 Hg*+>Cd*+
[351 Zn2+ Mgz+
E::;
anhydrase B
(a) Esterase activity (6) Hydration of CO* Glutamine synthetase (a) ADP reaction (6) AMP reaction Pyruvate
Inactive
aldolase
Concanavalin
Co2+>Zn2+>Cu2+>Mn2+ Ni2+>Cd2+ Zn2+>CoZ+>Ni2+>Cdz+
[371 Cl?+
1371
Cd2+>Mn2+>MgZ+>Ca2+ Coe+>Zng+ CdZ+>Mn2+
[381
Co2+>Ni2+>Zn2+>Cu2+ Mg2+>Ca2+
[391
[381
A
(a) Site 1
NiP+>Cd2+>CoZ+>ZnP+ >Mn2+ CaZ+>Cd2+>Sr2+
(6) Site 2
DISCUSSION This study presents evidence concerning the ionic requirements for cell attachment to collagen substrates. The influence of hydrogen ion concentration, monovalent, and multivalent cations on the process of cell attachment has been investigated. As indicated in fig. 1, the binding of cell attachment protein (c-CAP) to collagen occurs over a broad pH range. In contrast, the pH optimum for mammalian cell adhesion to a pre-formed c-CAP-collagen complex occurs in a relatively narrow physiological range. The inhibition of cell attachment observed at high pH may be explained by the formation of insoluble hydroxides of divalent cations [ 191. That divalent cations are involved in cellto-cell adhesion has been recognized since 1907 [23]. Over a period of years, Ca2+and/
Ca2+, MgZ+
1401
W+
[401
or Mg2+ have been demonstrated to be requirements in both cell re-aggregation phenomenon [24-261 and in several cell-to-substrate attachment systems [27-301. In the present investigation, it has been demonstrated that at least six divalent cations are effective in promoting cell attachment to collagen (table 1). Findings concerned with the role and specificity of metal ions in enzymatic catalysis are presented below for comparative purposes. The involvement of metal ions in cell attachment is by no means unique since at least one-third of the known enzymes are either activated by metal ions or contain a tightly bound metal ion [31, 321. The broad specificity of the cell adhesive response to divalent cations (table 1) finds parallels in enzymology ([32-341, for reviews). That many enzymes can be activated by more Exp Cell Res I10 (1977)
424
Klebe et al.
than one metal ion is indicated by the examples presented in table 2. At least twenty other enzymes also display a divalent metal ion requirement that can be sufficed by more than one element [33]. In these cases, the preference for one element over another depends on the enzyme studied (table 2). The relative order of preference of a given enzyme for one divalent cation over another can be varied by the pH of the assay medium [41] and the substrate employed [35-381. The discussion above thus indicates that (a) many enzymes can be activated by more than one divalent cation and (b) preference for one ion over another depends on the enzyme studied. The evidence presented indicates that Ca2+, Mg2+, and certain transition group elements can promote cell attachment to collagen (table 1). The ability of given transition group elements to replace Ca2+ and Mg2+ in this system appears to be physiologically relevant since (a) cell attachment protein (c-CAP) is required for response to the transition group metals (table 1) and (b) as in the case of Ca2+and Mg2+, the mutant CHOatt- cell line is markedly impaired in cell attachment to collagen promoted by transition group elements (table 1). Of those divalent cations studied, MnZ+ is most effective in promoting cell attachment while Ni2+ is the least effective (table 1). Polyamines and the large divalent cations, W+ and Ba2+, are inactive in this system (table 1). As previously pointed out, the requirement for and broad elemental specificity for divalent cations in cell attachment to collagen finds numerous parallels in enzymoh?Y. The mode of cell attachment mediated by Be2+, A12+,and Fe2+is probably not physiologically significant since (a) cells attached by the above ions are not dislodged by trypsin or EDTA, (b) CHO and the non-adheExp Cell Res 110 (1977)
sive mutant, CHOatt-, are equally responsive to these small multivalent cations and (c) cell attachment protein (c-CAP) is not required for attachment in the presence of this group of ions (table 1). The essentially irreversible, mode of cell attachment observed in the presence of Be2+, A13+, and Fe3+ may be accounted for by the high stability of the metal ion complexes formed by these small ions of high charge to radius ratio (Z/r) [43-45]. The finding that cell attachment can occur in the presence of isotonic NaCl, KCl, LiCl, NH,Cl, glucose and sucrose (fig. 2) indicates that no elemental specificity exists for the species of monovalent cation present during the process of cell attachment to collagen. In this regard, it might be noted that several enzymes do require monovalent cations for catalytic activity and that elemental specificity for monovalent cations is observed for certain enzymes [2022]. The observation that a wide variety of isotonic salts, or carbohydrates, can support cell attachment probably indicates that these agents function as osmotic stabilizing agents during cell attachment to collagen. That hypotonically swollen and hypertonitally crenated CHO cells are unable to attach to collagen implies that the interaction of the cell membrane with a c-CAP-collagen complex is not a simple lock-and-key reaction. Evidence indicating that the process of cell attachment requires metabolic energy [7, 251 also supports the view that more than a lock-and-key mechanism is involved in cell attachment to collagen. We thank MS Julie Machell for aid in preparation of the manuscript. These studies were supported, in part, by NIH grants CA 19017and GM 21433.
REFERENCES 1. Hausman, R E & Moscona, A A, Proc natl acad sci US 73 (1976) 3594.
Ionic requirements 2. Balsano, J & Lilien, J, Proc natl acad sci US 71 (1974) 727. 3. McClay, D R & Moscona, A A, Exp cell res 87 (1974) 438. 4. Henkart, P, Humphreys, S & Humphreys, T, Biochemistrv 12 (1973) 3045. 5. Pessac, B &*Defendi, V, Science 175 (1972) 898. 6. Klebe, R J, Nature 250 (1974) 248. - J cellular physiol86 (1975) 231. ii: Kleinman, H K, McGoodwin, E B & Klebe, R J, Biochem biophys res commun 72 (1976) 426. 9. Klebe, R J, Rosenberger, P G, Naylor, S L, Bums, R L, Novak, R 8t Kleinman, H, Exp cell res 104 (1977) 119. 10. Perlstein, E, Nature 262 (1976) 497. 11. Bull, H B, J colloid interface sci 41 (1972) 305. 12. Brash, J L L Lyman, D J, J biomed mater res 3 (1%9) 175. 13. Gabel, D, Steinberg, I Z & Katchalski, E, Biochemistrv 10 (1971) 4661. 14. Taylor, A C, Exp cell res, suppl. 8 (l%l) 154. 15. Unhjem, 0 & Prydy, H, Exp cell res 83 (1973) 418. 16. Takeda, Y & Oyiso, Y, FEBS lett 66 (1976) 332. 17. Peter, H W, Wolf, H U & Seiler, N, Hoppe Seyler’s Z physiol Chem 354 (1973) 1146. 18. Kao, F T &Puck, T T, Genetics 55 (1967) 513. 19. Weast, R C, Handbook of chemistry and physics, vol. 57. CRC Press, Cleveland, OH (197677). 20. Borden, R E & Scrutton, M C, J biol them 249 (1974) 4829. 21. Nowak, T, J biol them 248 (1973) 7191. 22. Avmch, J & Fairbanks, G, Biochemistry 13 (1974) 5507. 23. Wilson, H V, J exp zoo1 5 (1907) 245. 24. Zwilling, E, Science 120 (1954) 219. 25. McGuire, E J, J cell biol68 (1976) 90. 26. Homby, J E, J embryo1 exp morph 30 (1973) 511. 27. Veda, M J, Ito, T, Okada, T X & Ohnishi, J cell biol71 (1976) 670.
425
28. Grinnell, F, Exp cell res 97 (1976) 265. 29. Rabinovitch. M & DeStefano., M J. J cell biol 59 (1973) 165. ’ 30. Takeichi, M & Okada, T S, Exp cell res 74 (1972) 51. 31. Dixon, M & Webb, E C, Enzymes, 2nd edn, p. 672. Academic Press, New York (1964). 32. Mildvan, A S, The enzymes (ed PD Boyer) vol. 2, p. 445, 3rd edn. Academic Press, New York (1970). 33. Vallee, B L & Coleman, J E, Comprehen biochem 12 (1964) 165. 34. Eichhorh, G L (ed), Inorganic biochemistry (1973). Elsevier. New York (1975). 35. Coleman, J E & Vallee, B‘ L, J biol them 236 (l%l) 2244. 36. Cottam, G L, Kupiecki, F P & Coon, M J, J biol them 243 (1%8) 1630. Coleman, J E, Nature 214 (1%7) 193. 2 Hunt, J B, Smymiotis, P Z, Ginsburg, A & Stadtman, E R, Arch biochem biophys 166 (1975) 102. 39. Gallo, A A & Sable, H Z, Biochim biophys acta 302 (1973) 443. 40. Shoham, M, Kalb, A J & Pecht, I, Biochemistry 12 (1973) 1914. 41. Monder, C, Biochemistry 4 (1%5) 2677. 42. Mildvan. A S & Cohen, M., Adv enzvmol 33 (1970) 1.’ 43. Angelici, R J, Inorganic biochemistry (ed G L Eichhom) (1973) p. 63. Elsevier, New York (1975 edn). 44. Everest, D A, The chemistry of beryllium. Elsevier, New York (1964). 45. Cotton, F A &Wilkinson, G, Advanced inorganic chemistry. Interscience, New York (1966).
Received June 29, 1977 Accepted July 8, 1977
Exp Cell Res I10 (1977)
Experimental Cell Research 110 (1977) 419425
CELL
ATTACHMENT
TO COLLAGEN:
ROBERT J. KLEBE, JAMES R. HALL, Division
PAMELLA
THE IONIC
REQUIREMENTS
ROSENBERGER and W. DARYL DICKEY
of Human Genetics, Department of Human Biological Chemistry and Genetics, University of Texas Medical Blanch, Galveston, TX 77550, USA
SUMMARY This study is concerned with three aspects of the ionic requirements for cell attachment to collagen; namely (a) the divalent, (b) monovalent cation specificities and (c) pH optima for cell interaction with collagen. The divalent cation requirement for cell attachment to collagen can be sufliced by Ca*+, MgP+ and certain transition group metals; whereas Baz+, SrP+, and polyamines are inactive. The pH optimum for cell attachment in this system occurs in the physiological range. The monovalent cation requirement for cell attachment to collagen is satisfied by isotonic NaCl, KCl, LiCl, NH,Cl, sucrose, and glucose. Pronounced inhibition of cell attachment occurs under both hypertonic and hypotonic conditions.
Over the last few years, several laboratories cation requirement for cells to attach to the have demonstrated proteins that mediate c-CAP-collagen complex and (3) requirecell adhesion [l-lo]. This study is con- ment for cellular metabolic energy [6,7]. cerned with the ionic requirements of a The present investigation is concerned serum-derived collagen-dependent cell at- with three aspects of the ionic requirements tachment protein (c-CAP). Several aspects for the interaction of cells with a c-CAPof the mechanism of action of c-CAP have collagen complex, namely (a) divalent; (b) been previously described. Cell attachment monovalent specificities; and (c) pH optima to collagen requires (a) a high molecular for cell attachment. weight serum protein, (b) divalent cations, (c) an energy source (glucose) and (d) bufMETHODS AND MATERIALS fered saline [6,7]. The cell attachment pro- Maintenance of cell lines tein binds preferentially to the CB, cyano- In the experiments described below, CHO Chinese gen bromide peptide of the cY,-chain of type hamster ovary cells [18] and a mutant of CHO, termed CHOatt- [9], were employed. CHOatr- is defective in I collagen [8]. c-CAP binds to collagen in its adhesive response to purified c-CAP as well as to the absence of divalent cations; in contrast, both Ca*+ and MgP+ [9]. Due to the fact that CHO is a occurring L-proline auxotroph, growth medivalent cations are required for cultured naturally dium must either contain or be supplemented with cells to attach to a c-CAP-collagen complex L-proline [18]. Both the wild type and mutant CHO lines were routinely maintained with Dulbecco’s [6]. Hence, three steps in cell attachment to cell modified Eagle’s medium supplemented with 1 mM collagen can presently be identified; namely L-proline, 10% fetal calf serum (FCS), and 100 U/ml plus 100 &ml streptomycin. Cells were (1) binding of c-CAP to collagen (no di- penicillin maintained at 36S”c under a 10% CO,-in-air atmovalent cation requirement), (2) divalent sphere. Exp
Cell
Res
I10
(1977)
420
60
Klebe et al.
I
04 0
,
, 2
, 4
,
, 6
, 6
, 10
pH; ordinate: % cell attachment. pH optima for the process of cell attachment to collagen. The effect of pH on (a) binding of cell attachment protein (c-CAP) to collagen (X-X) and (b) cell attachment to a preformed c-CAP collagen complex (0-O) were studied as described in the text. The buffer ions which were employed at 10 mM were glycine OpH 1.85, pH 2.3, and pH 9.8), sodium acetate @H 4.8), MES @H 6.15), imidazole (PH 6.95), potassium phosphate (pH 7.2), Hepes @H 7.5), Bicine (PH 8.35), and borate (pH 9.2). As indicated above, c-CAP binds to collagen over a broad pH range while cell attachment to a pre-formed c-CAP-collagen complex takes place optimally in the physiological range.
Fig.
1. Abscissa:
Minimal attachment medium (MAM) A minimal attachment medium, termed MAM medium [6], has been employed in. the experiments described below. MAM medium contains 116 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 1 mM CaCl,, 1 mM MaCl,. and 10 mM Henes, DH 7.5. In exneriments co&erned with the requirements for divalent cations, Ca*+ and Mgl+ were either deleted or varied in most experiments. In studies concerned with the pH optimum for cell attachment, other buffer anions were employed, within 0.5 pH units of their respective pK’s, in place of Hepes. The buffer ions were tested at pH 7.5 in order to rule out any inhibitory effect of the buffer ion itself in pH optimal experiments. The buffers. which were emnloved (at 10 mM). are aiven in fg ‘1. In studies co&&ning the monovalent cation reauirement for cell attachment. the NaCl and KC1 components of MAM medium were either deleted or varied as described in the text.
Cell attachment to collagen assay The assay for cell attachment to collagen has been described in detail [6]. In brief, the assay procedure is as follows. c-CAP was permitted to bind to rat-tail collagen-coated bacteriological Petri plates in MAM medium. c-CAP purified to the 2nd PEG step [6] was employed. After an incubation period that permits c-CAP to bind to collagen, cultured cells were added to the c-CAP treated Petri plates and incubated at Exp Cell Res II0 (1977)
37°C for 1.5 h. At the end of the assay period, those cells that had attached were trypsinized and counted with an electronic cell counter. In the absence of c-CAP or an active divalent cation, no cell attachment to collagen is observed; in the presence of c-CAP and an active divalent cation, the number of cells attached is a function of the c-CAP or active divalent cation concentration [6]. In the studies described, the following technical modifications of the previously reported assay procedure [6] were employed. (1) Plates were not incubated in a CO, environment since the heavv metal ions studied are often precipitated as carbonates when CO, is oresent. (2) Since several oroteins 111-131and mammalian cells cl4, 151undergo-physical-adsorption to glass and plastic surfaces when exposure is carried out under protein-free conditions, Petri plates were pretreated with protein in order to avoid artifacts arising from physical adsorption. Thus, in experiments that involved deletion of protein from media, collagenized Petri plates were treated with 100 pg/ml bovine serum albumin (BSA) and washed three times with MAM medium (minus divalent cations). The BSA treatment renders the collagenized Petri plates wettable and minimizes the adsorption of other proteins and cells. (3) Cells were washed orior to the beginning of the assay with either (a) MA-M medium minus dc valent cations (in the case of experiments involving divalent cations); or (b) with MAM medium containing a single monovalent cation or carbohydrate at isotonic concentration (in the case of experiments involving monovalent cations).
Reagents All monovalent and divalent cations employed were in the chloride form. with the excention of Bex+ which was obtained as the sulfate. All chemicals employed were of reagent grade.
RESULTS pH Optimum for (a) cell attachment protein binding and(b) cell attachment to a c-CAP-collagen complex The pII optimum for cell attachment to collagen was determined by assessing the effect of pH on (a) the binding of c-CAP itself to collagen; (b) the attachment of cells to a pre-formed c-CAP-collagen complex. c-CAP is stable for at least 1 h in a range from pH 3 to 10.5. In order to determine the infhrence of pH on the binding of c-CAP to collagen, a saturating amount of purified c-CAP, equivalent in activity to 5 % calf serum, was ex-
421
Ionic requirements
Petri plates were (a) treated with a saturating amount of purified c-CAP at pH 7.5 in complete MAM medium: (b) washed twice with saline; (c) cells were then allowed to attach in modified MAM medium at PI-I’S established with the buffer ions given in the fig. 1. The pH optimum for cell attachment to a c-CAP-collagen complex was found to be narrower than the pH range in which c-CAP itself binds to collagen (fig. 1). 0
Fig.
001
063
2. Abscissa:
0’1
0’3
molar cont.;
ordinate:
%
cells at-
tached. Cell attachment to collagen was studied in the presence of several salts of monovalent cations or carbohydrates at the concentrations indicated above. The compounds tested for their ability to promote cell attachment were NaCl (0-O); KC1 (A-A); NH&l (O-O); LiCl (X-X); alucose (0-O): and sucrose (6-d). Above the &o&tic concentration, cells became crenated. Below the isotonic concentration, cells were microscooicallv observed to become increasinaly swollen as ihe salt or carbohydrate concentrati& was decreased. It should be noted that cell attachment was markedly inhibited at hypotonic concentrations that did not result in noticeable cell lysis. As indicated above, cell attachment to collagen is optimal at the isotonic concentration (about 0.15 M for salts and 0.3 M for carbohydrates).‘ Increasing inhibition of cell attachment occurs as the medium becomes more hypotonic or hypertonic.
posed to collagen-coated Petri plates at PI-I’S established with the buffer ions given in fig. 1. After a 1 h incubation at 37°C the c-CAP treated plates were (a) washed twice with saline to remove excess c-CAP, (b) washed with complete MAM medium (pH 7.5) and- (c) cell attachment was then assayed as described in the Methods. Under the conditions described above, c-CAP was found to bind to collagen between pH 1.95 to pH 9.8; however, maximum c-CAP binding occurred in the physiological range (fig. 1). In order to determine the pH range in which cells will bind to a pre-formed c-CAP-collagen complex, collagen-coated
Monovalent cation requirement for cell attachment to collagen
The monovalent cation requirement for cell attachment to a c-CAP-collagen complex wag studied
by (a)
treating
cofi~en-coated
Petri plates with a saturating level of purified c-CAP in complete MAM medium, (b) washing twice with MAM medium lacking monovalent cations and (c) adding CHO cells to c-CAP treated plates containing MAM
medium
modified
to include
only in-
dividual monovalent cations or carbohydrateg,
When employed
at or near their iso-
tonic concentrations, several monovalent cations and carbohydrates were capable of supporting cell attachment (fig. 2). Divalent cation requirement for cell attachment to collagen
We have previously shown that Ca2+ and/ or Mg2+ are required for cell attachment to collagen [6]. Several other di- and trivalent cations were tested for their ability to promote cell attachment to collagen. Those multivalent cations that formed precipitates at or below 1 mM were excluded from the study. The cations studied are presented in an order of increasing ionic radius in table 1 and have been divided into three classes on the basis of their cell attachment properties. Exp Cell Res 110 (1977)
422
Klebe et al.
Table 1. Di- and trivalent cation requirement for 50 % cell attachment to collagen Divalent cation”
Crystal ionic radius b
ClassI BePfd AP+ d Fe3+d
0.35 A
5.7
0.51 0.64
5.8 4.6
Class ZZ MiP+ Ni2+ co*+ Zd+ Mn2+ Ca2+
0.66A 0.69 0.72 0.74 0.80 0.99
3.0 2.9
Class III se+ Ba2+
CHOa”-
CHO Z/R Ratioc
+ c-CAP
3x10-*
:.:: 2:5 2.0
1.12A
1.8
1.34
1.5
-c-CAP
+c-CAP
-c-CAP
6x lo+ mM
3x 1OV mM 10-Z ax 10-S
3x lo-* mM
8X 10-Z
>lOmM > 10e >lO > 10e >lO >lO
3mM >lO’ 1 >1oe 3x 10-Z 100
>lOmM > 10” >lO > 1oe >lO >lO
>lO mM >lO
>lOmM >lO
>lOmM >lO
>lO mM >lO
mM
10-Z 10-Z
10-Z 10-Z
lo-* mM 2x10-1 6x lo+
10-l
8x lo+
10-Z 10-Z
Cell attachment assays were carried out as described in the text. Collagen coated Petri plates were treated with 100 &ml BSA for 30 min and washed twice with MAM (minus divalent cations) prior to use in the assay. Minimal Attachment Medium (MAM) minus divalent cations was employed for washing cells. In the case of those divalent cations that promoted cell attachment to collagen, the concentration required for 50 % cell attachment is entered. a Multivalent cations which formed precipitates at or below 1 mM were excluded from the study. b Crystal ionic radii were taken from [ 191. c Z/R is the charge to radius ratio. d Cells cannot be removed by trypsin. e Precipitate formed at 10 mM.
Class I. The di- and trivalent ions in class I are smaller in ionic radius than MgZ+ and were found to attach cells in the presence or absence of c-CAP (table 1). In addition, ions of class I avidly promoted the attachment of a mutant (CHOati-) that is defective in cell attachment to collagen [9] (table 1). Cells attached to collagen by ions in class I were found to be resistant to dislodgement from collagen by both trypsin and EDTA. Cells attached to collagen by 0.1 mM Be2+ were found to resist removal from collagen for at least 16 h by 1000 times the trypsin concentration required to remove Ca2+-attached cells in 15 min. Similar results were obtained in the case of A13+and Fe3+. Class ZZ. The divalent cations in class II range in ionic radius between that of Mg2+ and Ca2+. Divalent cations of class II reExp CellResIIO (1977)
quired c-CAP in order to attach cells to collagen (table 1). As is indicated in table 1, CHOatt-, which is genetically altered in its response to Ca2+and Mg2+ [9], was also defective in its response to Ni2+, Co2+, Zn2+, and Mn2+. Class ZZZ. Divalent cations in class III are larger in ionic radius than Ca2+. These ions did not promote cell attachment to collagen (table 1). Polyamines. Since the divalent metal ion requirement of certain enzymes can be sufficed by polyamines [16, 171, several multivalent amino compounds were investigated as replacements for metal ions in the cell attachment process. At concentrations as high as 10 mM, hydrazine, ethylenediamine, putrescine, and spermidine were not able to substitute for divalent metal ions.
Ionic requirements
423
Table 2. Divalent cation activation and inhibition of enzymes Enzyme
Activators
Carboxypeptidase A (a) Esterase activity
Inhibitors
(6) Peptidase activity
Cde+>Hg*+>Zn2+>Co2+ >Ni2+>MnZ+ CoZ+>Ni2+>ZnZ+>Mn*+
Pyruvate kinase (a) Pyruvate reaction (6) Hydroxylamine reaction
Mg*+, Mn*+ Co2+>ZnZ+>Mnz+
Carbonic
Ref.
1351 Hg*+>Cd*+
[351 Zn2+ Mgz+
E::;
anhydrase B
(a) Esterase activity (6) Hydration of CO* Glutamine synthetase (a) ADP reaction (6) AMP reaction Pyruvate
Inactive
aldolase
Concanavalin
Co2+>Zn2+>Cu2+>Mn2+ Ni2+>Cd2+ Zn2+>CoZ+>Ni2+>Cdz+
[371 Cl?+
1371
Cd2+>Mn2+>MgZ+>Ca2+ Coe+>Zng+ CdZ+>Mn2+
[381
Co2+>Ni2+>Zn2+>Cu2+ Mg2+>Ca2+
[391
[381
A
(a) Site 1
NiP+>Cd2+>CoZ+>ZnP+ >Mn2+ CaZ+>Cd2+>Sr2+
(6) Site 2
DISCUSSION This study presents evidence concerning the ionic requirements for cell attachment to collagen substrates. The influence of hydrogen ion concentration, monovalent, and multivalent cations on the process of cell attachment has been investigated. As indicated in fig. 1, the binding of cell attachment protein (c-CAP) to collagen occurs over a broad pH range. In contrast, the pH optimum for mammalian cell adhesion to a pre-formed c-CAP-collagen complex occurs in a relatively narrow physiological range. The inhibition of cell attachment observed at high pH may be explained by the formation of insoluble hydroxides of divalent cations [ 191. That divalent cations are involved in cellto-cell adhesion has been recognized since 1907 [23]. Over a period of years, Ca2+and/
Ca2+, MgZ+
1401
W+
[401
or Mg2+ have been demonstrated to be requirements in both cell re-aggregation phenomenon [24-261 and in several cell-to-substrate attachment systems [27-301. In the present investigation, it has been demonstrated that at least six divalent cations are effective in promoting cell attachment to collagen (table 1). Findings concerned with the role and specificity of metal ions in enzymatic catalysis are presented below for comparative purposes. The involvement of metal ions in cell attachment is by no means unique since at least one-third of the known enzymes are either activated by metal ions or contain a tightly bound metal ion [31, 321. The broad specificity of the cell adhesive response to divalent cations (table 1) finds parallels in enzymology ([32-341, for reviews). That many enzymes can be activated by more Exp Cell Res I10 (1977)
424
Klebe et al.
than one metal ion is indicated by the examples presented in table 2. At least twenty other enzymes also display a divalent metal ion requirement that can be sufficed by more than one element [33]. In these cases, the preference for one element over another depends on the enzyme studied (table 2). The relative order of preference of a given enzyme for one divalent cation over another can be varied by the pH of the assay medium [41] and the substrate employed [35-381. The discussion above thus indicates that (a) many enzymes can be activated by more than one divalent cation and (b) preference for one ion over another depends on the enzyme studied. The evidence presented indicates that Ca2+, Mg2+, and certain transition group elements can promote cell attachment to collagen (table 1). The ability of given transition group elements to replace Ca2+ and Mg2+ in this system appears to be physiologically relevant since (a) cell attachment protein (c-CAP) is required for response to the transition group metals (table 1) and (b) as in the case of Ca2+and Mg2+, the mutant CHOatt- cell line is markedly impaired in cell attachment to collagen promoted by transition group elements (table 1). Of those divalent cations studied, MnZ+ is most effective in promoting cell attachment while Ni2+ is the least effective (table 1). Polyamines and the large divalent cations, W+ and Ba2+, are inactive in this system (table 1). As previously pointed out, the requirement for and broad elemental specificity for divalent cations in cell attachment to collagen finds numerous parallels in enzymoh?Y. The mode of cell attachment mediated by Be2+, A12+,and Fe2+is probably not physiologically significant since (a) cells attached by the above ions are not dislodged by trypsin or EDTA, (b) CHO and the non-adheExp Cell Res 110 (1977)
sive mutant, CHOatt-, are equally responsive to these small multivalent cations and (c) cell attachment protein (c-CAP) is not required for attachment in the presence of this group of ions (table 1). The essentially irreversible, mode of cell attachment observed in the presence of Be2+, A13+, and Fe3+ may be accounted for by the high stability of the metal ion complexes formed by these small ions of high charge to radius ratio (Z/r) [43-45]. The finding that cell attachment can occur in the presence of isotonic NaCl, KCl, LiCl, NH,Cl, glucose and sucrose (fig. 2) indicates that no elemental specificity exists for the species of monovalent cation present during the process of cell attachment to collagen. In this regard, it might be noted that several enzymes do require monovalent cations for catalytic activity and that elemental specificity for monovalent cations is observed for certain enzymes [2022]. The observation that a wide variety of isotonic salts, or carbohydrates, can support cell attachment probably indicates that these agents function as osmotic stabilizing agents during cell attachment to collagen. That hypotonically swollen and hypertonitally crenated CHO cells are unable to attach to collagen implies that the interaction of the cell membrane with a c-CAP-collagen complex is not a simple lock-and-key reaction. Evidence indicating that the process of cell attachment requires metabolic energy [7, 251 also supports the view that more than a lock-and-key mechanism is involved in cell attachment to collagen. We thank MS Julie Machell for aid in preparation of the manuscript. These studies were supported, in part, by NIH grants CA 19017and GM 21433.
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28. Grinnell, F, Exp cell res 97 (1976) 265. 29. Rabinovitch. M & DeStefano., M J. J cell biol 59 (1973) 165. ’ 30. Takeichi, M & Okada, T S, Exp cell res 74 (1972) 51. 31. Dixon, M & Webb, E C, Enzymes, 2nd edn, p. 672. Academic Press, New York (1964). 32. Mildvan, A S, The enzymes (ed PD Boyer) vol. 2, p. 445, 3rd edn. Academic Press, New York (1970). 33. Vallee, B L & Coleman, J E, Comprehen biochem 12 (1964) 165. 34. Eichhorh, G L (ed), Inorganic biochemistry (1973). Elsevier. New York (1975). 35. Coleman, J E & Vallee, B‘ L, J biol them 236 (l%l) 2244. 36. Cottam, G L, Kupiecki, F P & Coon, M J, J biol them 243 (1%8) 1630. Coleman, J E, Nature 214 (1%7) 193. 2 Hunt, J B, Smymiotis, P Z, Ginsburg, A & Stadtman, E R, Arch biochem biophys 166 (1975) 102. 39. Gallo, A A & Sable, H Z, Biochim biophys acta 302 (1973) 443. 40. Shoham, M, Kalb, A J & Pecht, I, Biochemistry 12 (1973) 1914. 41. Monder, C, Biochemistry 4 (1%5) 2677. 42. Mildvan. A S & Cohen, M., Adv enzvmol 33 (1970) 1.’ 43. Angelici, R J, Inorganic biochemistry (ed G L Eichhom) (1973) p. 63. Elsevier, New York (1975 edn). 44. Everest, D A, The chemistry of beryllium. Elsevier, New York (1964). 45. Cotton, F A &Wilkinson, G, Advanced inorganic chemistry. Interscience, New York (1966).
Received June 29, 1977 Accepted July 8, 1977
Exp Cell Res I10 (1977)
