Brilish fourmi
of Derniatohfui
( 1 9 9 2 ) 1 2 6 . 2 3 1-2 3 b .
Increased calmodulin levels in fibroblasts from progressive systemic sclerosis M.SEISHIMA. S.NAGAO.* M.KUWAHARA. S.MORI AND Y.NOZAWA* Department of Dermatology and *Btochemistry. Gifu University School of Medicine. Gifu. lapan Accepted for publication Ifi OitobtT 1991
Summary
Calmodulin levels in cultured skin fibroblasts from patients with progressive systemic sclerosis (PSS) and healthy controls were tneasured by their ability to activate cyclic AMP-phosphodiesterase. Calmodulin levels were significantly increased in PSS fibroblasts compared with normal control fibroblasts. The changes in calmodulin content of PSS tibroblasts were also assessed by a radioimmunoassay. These findings suggest that an elevated level of calmodulin may play a role in the pathogenesis of PSS.
Progressive systemic sclerosis (PSS) is a connective tissue disease characterized by excessive deposition of collagen and other connective tissue components in the skin and internal organs. Although manifestations of the disease have been reported to depend on a complex interplay of genetic factors.' immunological abnormalitie.s.' microvascular responses* and connective tissue cell activation.'^ fibroblasts are a likely candidate for studies designed to examine possible tnetabolic defect(s) that may be associated with PSS. PSS fibroblasts appear to have many unusual properties. Cultivation of PSS tibroblasts in the presence of 10% foetal calf serum resulted in an increased population-doubling time over controls. "^ An itnpairment was reported of PSS fibroblast sensitivity to lyniphoid cell-derived fibroblast growth factor (FC.F) and platelet-derived fibroblast growth factor (PD(iI'). In addition, the synthesis of structural proteins such as collagen, fibronectin and glycosaminoglycan. known to be a major function of fibroblasts during development and differentiation/'^ is increased in PSS libroblasts.'"-'Calcium and its binding protein caimodulin are known to modulate proliferation, differentiation and metabolism in a variety of cell types."''^ Calmodulin is an acidic protein with a molecular weight of 16.7(K) which modulates the actions of calcium. For example, calmodulin is known to associate with mitotic spindles during cell division'' and its inhibitors suppress proliferation of cells in vitro.^'^ It was also observed that the level of calmodulin is elevated in cells showing abnormal proliferation, such as transformed cell lines of Swiss 3T5 Correspondence: Dr Mariko Seishima. Department of Demiatology, C.ifu University School of Medicine. Tsukasamachi-4(), Oifii 50(1, lapan.
cells,'' chicken embryo fibroblasts,'" hepatoma cells'"* and psoriatic keratinocytes."" '- To investigate whether calmodulin is implicated in the pathogenesis of PSS. we have compared the calmodulin levels in cultured fibroblasts from PSS patients and healthy controls.
Methods Materials
Eagle's tiiinimum essential medium (MEM) and glutamine were purchased from Nissui Pharmaceutical Co. (Tokyo, lapan). and foetal calf serum from Irvine Scientific (Santa Ana. CA. U.S.A). Cyclic AMP (cAMP) and 5'-nucleotidase {Crotaius atrox venom) were from Sigma Chemical Co. (St Louis. MO. U.S.A.!. Cahnodulin radioimtnunoassay kits were obtained from New Hngland Nuclear (Boston. MA, U.S.A.). The calmodulin standard was extracted from rat brain by the trichloroacetic acid-precipitation method and then purified.'' Calmodulin-deficient cAMP-phosphodiesterase was prepared from rat brain by successive chromatographies on DEAE-celluiose and Sephadex G-200 columns.-^ All other chemicals were of reagent grade. Fibroblast culture
Skin samples were obtained from the forearms of normal healthy controls atid from patients with PSS (Table 1). All the PSS patients had diffuse scleroderma with skin lesions on the extremities and the fingers, hands and face (classical scleroderma). None of the patients were on systemic steroids, cytotoxic drugs or other drugs such as penicillamine that might affect fibroblast proliferation. Each skin biopsy was immediately rinsed three titnes 231
M.SEISHIMA el ul.
Table 1. Quantitation of calmodulin levels in PSS and normal iibroblasts
Case PSS 1 2 3 4 5 6 7 8
Age
t.) Lira I ion
riiidiitgs in
Sex
(years)
(yearsi
biupsied area
Medication
F F
44 38 63 44 54 59 35 11
3 10 2 4 6 10 5 10
Oedema Sclerosis Sclerosis Oedema, sclerosis Sclerosis Sclerosis Sclerosis Sclerosis
Vitamin C, E None Vimatin t:: Vilamin C
M F F F
F F
Calmodulin* (U/mg/ protein)
Caimodulint t/ig/mg protein)
16-0 28 8 36-4 45-9 47 3
0-67 0-83 1 01 0 96 1-59
504 ND ND
098 IJ9
None None None None
3 7 S ± n 2'
Total
1-72 1 1 4 ± 0 38**
Controls I 2 3 4
5 6 7 8
V M M F F M F F
4-5
42 19 60 59 37 28 52 48
13 4 16-5 17-6 21-3 22-7 ND
ND 16-()±6-6'
T o t ill
0 31 0 45 t)39 042 049
0-58 0 79 1 02 t) 5f)±()24"
* Extracts cnnlaining calmodulin were prepared as described in Methods. One unit of talniodulin was detined as the amount givtng half-maximal stlmuliitiiin of the cAMP-phosphtKiiestertise. t Oilruidulin amtt-nt was determined by radioimmunoassay. •/' Mfd 1979: 149: 1 32f)-J5. 4 Buckingham RB. Prince PK. Rodnan GP. Taylor F. Increased colliigen at cumulation in demial fibroblast cultures from p(itient.s with prof^ressive syslemic sclerosis (sclcrodcrma). / 1-ah Clin Mi'd 1978:92: 5-21. 5 Kovacs C], Fleischmaier R. Properties of scleroderma fibrobiasts In culture. / /7iW5( Deniuitol 1974; 63: 45fi-fi(). (S URoy \iC. Mercurio S, Shcrcr GK. Repliaition and phcnotypic expression of conlrol and sd erode nil a human fibroblasts. Kcsponsfs to growth factor. I'roc Sull Ami Sci 19S2: 79: Ii8(i-9U. 7 Nishiokci N, Kobayashi V. K»Uiyva Ml). Kbokblova |V, Pananyuk cl «/. Cyclic nucleotides and caicium transpori in cultured demiai fibroblasts from progressive systemic sclerosis and rbeumatoid arthritis patients. Arthritis Rheum 1984: 27: 1144-9. 29 Rasmussen CD. Means AR.Calmcdulin is involvt-d in regulation of cell proliferation. EMBO I !987: 13: J96I-8. 30 Seis!iima M. Yada Y. Nagao S el al. Defective formation of inositol 1.4,5-trijip!iosphale in bradykinin-stimulated fibroblasts from progressive systemic sclerotic patients. BiorlK'n\ liioi>hiis Res Cominuii 1988: t56: 1077-82. Jl Scisliimci M. Kudo Y. Nagao S ft al. Alterations in intraceilular calcium transients of Hbroblasts from progressive systemic sclerotic patients: a digitai irnaging microscopic study. /1rc/i Derimitoi Res !99I: 283: 96-9. 32 Wolff i)l. Brostrom CO. Properties and functions of the calciumdependent rcguiator protein. Adv Cyclic Nueleotide Res 1979: 11: 27-88.
of Derniatohfui
( 1 9 9 2 ) 1 2 6 . 2 3 1-2 3 b .
Increased calmodulin levels in fibroblasts from progressive systemic sclerosis M.SEISHIMA. S.NAGAO.* M.KUWAHARA. S.MORI AND Y.NOZAWA* Department of Dermatology and *Btochemistry. Gifu University School of Medicine. Gifu. lapan Accepted for publication Ifi OitobtT 1991
Summary
Calmodulin levels in cultured skin fibroblasts from patients with progressive systemic sclerosis (PSS) and healthy controls were tneasured by their ability to activate cyclic AMP-phosphodiesterase. Calmodulin levels were significantly increased in PSS fibroblasts compared with normal control fibroblasts. The changes in calmodulin content of PSS tibroblasts were also assessed by a radioimmunoassay. These findings suggest that an elevated level of calmodulin may play a role in the pathogenesis of PSS.
Progressive systemic sclerosis (PSS) is a connective tissue disease characterized by excessive deposition of collagen and other connective tissue components in the skin and internal organs. Although manifestations of the disease have been reported to depend on a complex interplay of genetic factors.' immunological abnormalitie.s.' microvascular responses* and connective tissue cell activation.'^ fibroblasts are a likely candidate for studies designed to examine possible tnetabolic defect(s) that may be associated with PSS. PSS fibroblasts appear to have many unusual properties. Cultivation of PSS tibroblasts in the presence of 10% foetal calf serum resulted in an increased population-doubling time over controls. "^ An itnpairment was reported of PSS fibroblast sensitivity to lyniphoid cell-derived fibroblast growth factor (FC.F) and platelet-derived fibroblast growth factor (PD(iI'). In addition, the synthesis of structural proteins such as collagen, fibronectin and glycosaminoglycan. known to be a major function of fibroblasts during development and differentiation/'^ is increased in PSS libroblasts.'"-'Calcium and its binding protein caimodulin are known to modulate proliferation, differentiation and metabolism in a variety of cell types."''^ Calmodulin is an acidic protein with a molecular weight of 16.7(K) which modulates the actions of calcium. For example, calmodulin is known to associate with mitotic spindles during cell division'' and its inhibitors suppress proliferation of cells in vitro.^'^ It was also observed that the level of calmodulin is elevated in cells showing abnormal proliferation, such as transformed cell lines of Swiss 3T5 Correspondence: Dr Mariko Seishima. Department of Demiatology, C.ifu University School of Medicine. Tsukasamachi-4(), Oifii 50(1, lapan.
cells,'' chicken embryo fibroblasts,'" hepatoma cells'"* and psoriatic keratinocytes."" '- To investigate whether calmodulin is implicated in the pathogenesis of PSS. we have compared the calmodulin levels in cultured fibroblasts from PSS patients and healthy controls.
Methods Materials
Eagle's tiiinimum essential medium (MEM) and glutamine were purchased from Nissui Pharmaceutical Co. (Tokyo, lapan). and foetal calf serum from Irvine Scientific (Santa Ana. CA. U.S.A). Cyclic AMP (cAMP) and 5'-nucleotidase {Crotaius atrox venom) were from Sigma Chemical Co. (St Louis. MO. U.S.A.!. Cahnodulin radioimtnunoassay kits were obtained from New Hngland Nuclear (Boston. MA, U.S.A.). The calmodulin standard was extracted from rat brain by the trichloroacetic acid-precipitation method and then purified.'' Calmodulin-deficient cAMP-phosphodiesterase was prepared from rat brain by successive chromatographies on DEAE-celluiose and Sephadex G-200 columns.-^ All other chemicals were of reagent grade. Fibroblast culture
Skin samples were obtained from the forearms of normal healthy controls atid from patients with PSS (Table 1). All the PSS patients had diffuse scleroderma with skin lesions on the extremities and the fingers, hands and face (classical scleroderma). None of the patients were on systemic steroids, cytotoxic drugs or other drugs such as penicillamine that might affect fibroblast proliferation. Each skin biopsy was immediately rinsed three titnes 231
M.SEISHIMA el ul.
Table 1. Quantitation of calmodulin levels in PSS and normal iibroblasts
Case PSS 1 2 3 4 5 6 7 8
Age
t.) Lira I ion
riiidiitgs in
Sex
(years)
(yearsi
biupsied area
Medication
F F
44 38 63 44 54 59 35 11
3 10 2 4 6 10 5 10
Oedema Sclerosis Sclerosis Oedema, sclerosis Sclerosis Sclerosis Sclerosis Sclerosis
Vitamin C, E None Vimatin t:: Vilamin C
M F F F
F F
Calmodulin* (U/mg/ protein)
Caimodulint t/ig/mg protein)
16-0 28 8 36-4 45-9 47 3
0-67 0-83 1 01 0 96 1-59
504 ND ND
098 IJ9
None None None None
3 7 S ± n 2'
Total
1-72 1 1 4 ± 0 38**
Controls I 2 3 4
5 6 7 8
V M M F F M F F
4-5
42 19 60 59 37 28 52 48
13 4 16-5 17-6 21-3 22-7 ND
ND 16-()±6-6'
T o t ill
0 31 0 45 t)39 042 049
0-58 0 79 1 02 t) 5f)±()24"
* Extracts cnnlaining calmodulin were prepared as described in Methods. One unit of talniodulin was detined as the amount givtng half-maximal stlmuliitiiin of the cAMP-phosphtKiiestertise. t Oilruidulin amtt-nt was determined by radioimmunoassay. •/' Mfd 1979: 149: 1 32f)-J5. 4 Buckingham RB. Prince PK. Rodnan GP. Taylor F. Increased colliigen at cumulation in demial fibroblast cultures from p(itient.s with prof^ressive syslemic sclerosis (sclcrodcrma). / 1-ah Clin Mi'd 1978:92: 5-21. 5 Kovacs C], Fleischmaier R. Properties of scleroderma fibrobiasts In culture. / /7iW5( Deniuitol 1974; 63: 45fi-fi(). (S URoy \iC. Mercurio S, Shcrcr GK. Repliaition and phcnotypic expression of conlrol and sd erode nil a human fibroblasts. Kcsponsfs to growth factor. I'roc Sull Ami Sci 19S2: 79: Ii8(i-9U. 7 Nishiokci N, Kobayashi V. K»Uiyva Ml). Kbokblova |V, Pananyuk cl «/. Cyclic nucleotides and caicium transpori in cultured demiai fibroblasts from progressive systemic sclerosis and rbeumatoid arthritis patients. Arthritis Rheum 1984: 27: 1144-9. 29 Rasmussen CD. Means AR.Calmcdulin is involvt-d in regulation of cell proliferation. EMBO I !987: 13: J96I-8. 30 Seis!iima M. Yada Y. Nagao S el al. Defective formation of inositol 1.4,5-trijip!iosphale in bradykinin-stimulated fibroblasts from progressive systemic sclerotic patients. BiorlK'n\ liioi>hiis Res Cominuii 1988: t56: 1077-82. Jl Scisliimci M. Kudo Y. Nagao S ft al. Alterations in intraceilular calcium transients of Hbroblasts from progressive systemic sclerotic patients: a digitai irnaging microscopic study. /1rc/i Derimitoi Res !99I: 283: 96-9. 32 Wolff i)l. Brostrom CO. Properties and functions of the calciumdependent rcguiator protein. Adv Cyclic Nueleotide Res 1979: 11: 27-88.
