Calcium-Dependent Signaling in Dupuytren's Disease

Abstract

Background

Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility. Cell contractility in smooth muscle cells is caused by calcium-dependent and calcium-independent signaling mechanisms. In the calcium-dependent pathway, calcium/calmodulin activates myosin light chain kinase (MLCK). In this study, the effects of calcium/calmodulin inhibition with the FDA-approved drug fluphenazine on Dupuytren's fibroblast contractility and MLCK expression were tested.

Methods

Fibroblast lines from the palmar fascia of patients with Dupuytren's disease were explanted and used for in vitro study. The effect of fluphenazine on Dupuytren's fibroblast migration was determined using a scratch migration assay, and contractility was determined using fibroblast-populated collagen lattice (FPCL) assays. Immunohistochemical staining of MLCK in different samples of Dupuytren's tissue and normal fascia were compared.

Results

Fluphenazine demonstrated a dose-dependent inhibition of Dupuytren's fibroblast migration, with the maximum inhibition of migration observed at 20 μM (69.8±1.9%). Fluphenazine also inhibited FPCL contraction in a dose-dependent manner. Maximal inhibition was observed at a fluphenazine concentration of 20 μM (52.5±6.1%). Immunohistological staining illustrated that MLCK was predominantly expressed throughout the cytoplasm of select fibroblasts within Dupuytren's nodules, yet was absent in the fibroblasts of Dupuytren's cords and normal palmar fascia.

Conclusions

Fluphenazine inhibits Dupuytren's fibroblast contractility and migration through inhibition of MLCK in vitro. However, the inconsistent expression of MLCK throughout Dupuytren's tissue suggests that calcium-dependent signaling may not be a primary mode of contracture formation. Fluphenazine inhibition of MLCK is not likely to be a target for the treatment of Dupuytren's disease.

Keywords

Calcium-dependent signaling

Get full access to this article

View all access options for this article.

References

Altamura

Sassella

Santini

. Intramuscular preparations of antipsychotics: uses and relevance in clinical practice. Drugs. 2003;63:493–512.

Altamura

Whelpton

Curry

. Animal model for investigation of fluphenazine kinetics after administration of long-acting esters. Biopharm Drug Dispos. 1979;1:65–72.

Anderson

DiCesare

Tan

. Rho-mediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts. Exp Cell Res. 2004;298:574–83.

Arafa

Noble

Royle

. Dupuytren's and epilepsy revisited. J Hand Surg (Br). 1992;17:221–4.

Burridge

Chrzanowska-Wodnicka

. Focal adhesions, contractility, and signaling. Ann Rev Cell Dev Biol. 1996;12:463–518.

Caroli

Zanasi

Marcuzzi

. Epidemiological and structural findings supporting the fibromatous origin of dorsal knuckle pads. J Hand Surg (Br). 1991;16:258–62.

Darby

Skalli

Gabbiani

. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest. 1990;63:21–9.

Davis

Leor

Watanabe

. Depot antipsychotic drugs, place in therapy. Drugs. 1994;47:741–73.

Gabbiani

Majno

. Dupuytren's contracture: fibroblast contraction? An ultrastructural study. Am J Pathol. 1972;66:131–46.

10.

Ganitkevich

Hasse

Pfitzer

. Ca2+-dependent and Ca2+-independent regulation of smooth muscle contraction. J Muscle Res Cell Motil. 2002;23:47–52.

11.

Gao

Kishi

. Myosin light chain kinase as a multifunctional regulatory protein of smooth muscle contraction. IUBMB Life. 2001;51:337–44.

12.

Goetz

Tanner

Klawans

. Fluphenazine and multifocal tic disorders. Arch Neurol. 1984;41:271–2.

13.

Grasso

Lania

Blanco

. The natural history of Peyronie's disease. Arch Esp Urol. 2007;60:326–31.

14.

Gudmundsson

Arngrimsson

Jonsson

. Dupuytren's disease, alcohol consumption and alcoholism. Scand J Prim Health Care. 2001;19:186–90.

15.

Gudmundsson

Arngrimsson

Jonsson

. Eighteen years follow-up study of the clinical manifestations and progression of Dupuytren's disease. Scand J Rheumatol. 2001;30:31–4.

16.

Gudmundsson

Arngrimsson

Sigfusson

. Epidemiology of Dupuytren's disease: clinical, serological, and social assessment. The Reykjavik Study. J Clin Epidemiol. 2000;53:291–6.

17.

Haedicke

Sturim

. Plantar fibromatosis: an isolated disease. Plast Reconstr Surg. 1989;83:296–300.

18.

Hindocha

John

Stanley

. The heritability of Dupuytren's disease: familial aggregation and its clinical significance. J Hand Surg (Am). 2006;31:204–10.

19.

Hindocha

Stanley

Watson

. Dupuytren's diathesis revisited: evaluation of prognostic indicators for risk of disease recurrence. J Hand Surg (Am). 2006;31:1626–34.

20.

Kamm

Stull

. Regulation of smooth muscle contractile elements by second messengers. Annu Rev Physiol. 1989;51:299–313.

21.

Kamm

Stull

. Dedicated myosin light chain kinases with diverse cellular functions. J Biol Chem. 2000;276:4527–30.

22.

Knobloch

Redeker

Vogt

. Antifibrotic medication using a combination of N-acetyl-L-cystein (NAC) and ACE inhibitors can prevent recurrence of Dupuytren's disease. Med Hypotheses. 2009;73:659–61.

23.

Kuhn

Wang

Payne

. Tamoxifen decreases fibroblast function and downregulates TGFβ2 in Dupuytren's affected palmar fascia. J Surg Res. 2002;103:146–52.

24.

Lau

Gnegy

. Effects of lanthanum and trifluoperazine on [125I] calmodulin binding to rat striatal particulates. J Pharmacol Exp Ther. 1980;215:28–34.

25.

Levin

Weiss

. Inhibition by trifluoperazine of calmodulin-induced activation of ATPase activity of rat erythrocyte. Neuropharmacology. 1980;19:169–74.

26.

Levinson

Moyer

Saggers

. Calmodulin-myosin light chain kinase inhibition changes fibroflast-populated collagen lattice contraction, cell migration, focal adhesion formation, and wound contraction. Wound Repair Regen. 2004;12:505–11.

27.

Ling

. The genetic factor in Dupuytren's disease. J Bone Joint Surg (Br). 1963;45:709–18.

28.

Naumanen

Lappalainen

Hotulainen

. Mechanisms of actin stress fibre assembly. J Microsc. 2008;231:446–54.

29.

Parizi

Howard

Tomasek

. Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase. Exp Cell Res. 2000;254:210–20.

30.

Pavelic

Bratulic

Hock

. Screening of potential prodrugs on cells derived from Dupuytren's disease patients. Biomed Pharmacother. 2009;63:577–85.

31.

Pfitzer

. Invited review: regulation of myosin phosphorylation in smooth muscle. J Appl Physiol. 2001;91:497–503.

32.

Rayan

. Dupuytren's disease: anatomy, pathology, presentation, and treatment. Instr Course Lect. 2007;56:101–11.

33.

Rayan

Tomasek

. Generation of contractile force by cultured Dupuytren's disease and normal palmar fibroblasts. Tissue Cell. 1994;26(5):747–56.

34.

Ross

. Epidemiology of Dupuytren's disease. Hand Clin. 1999;15:53–62.

35.

Sandquist

Swenson

Demali

. Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration. J Biol Chem. 2006;281(47):35873–83.

36.

Sellers

Wang

Chantler

. Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins. J Muscle Res Cell Motil. 2003;24(8):579–85.

37.

Stull

Lin

Krueger

. Myosin light chain kinase: functional domains and structural motifs. Acta Physiol Scand. 1998;164:471–82.

38.

Tomasek

Gabbiani

Hinz

. Myofibroblasts and mechano-regulation of connective tissue remodeling. Nat Rev Mol Cell Biol. 2002;3:349–63.

39.

Tomasek

Haakama

Eddy

. Fibroblast contraction occurs on release of tension in attached collagen lattices: dependency on an organized actin cytoskeleton and serum. Anat Rec. 1992;232:359–68.

40.

Tomasek

Rayan

. Correlation of alpha-smooth muscle actin expression and contraction in Dupuytren's disease fibroblasts. J Hand Surg (Am). 1995;20(3):450–5.

41.

Tomasek

Vaughan

Haaksma

. Cellular structure and biology of Dupuytren's disease. Hand Clin. 1999;15:21–34.

42.

Townley

Cambrey

Khaw

. Matrix metalloproteinase inhibition reduces contraction by Dupuytren fibroblasts. J Hand Surg Am. 2008;33:1608–16.

43.

Vincenzi

. Calmodulin pharmacology. Cell Calcium. 1981;2:387–409.

44.

Weiss

Levin

. Mechanism for selectively inhibiting the activation of cyclic nucleotide phophodiesterase and adenylate cyclase by antipsychotic agents. Adv Cyclic Nucleotide Res. 1978;9:285–303.

45.

Zachariae

. Dupuytren's contracture. The aetiological role of trauma. Scand J Plast Reconstr Surg. 1971;5:116–9.