Creatine Kinase and Creatine Transporter in Normal, Wounded, and Diseased Skin
2002; Elsevier BV; Volume: 118; Issue: 3 Linguagem: Inglês
10.1046/j.0022-202x.2001.01697.x
ISSN1523-1747
AutoresUwe Schlattner, Natalie Möckli, Oliver Speer, Sabine Werner, Theo Wallimann,
Tópico(s)Body Contouring and Surgery
ResumoSkin comprises many cell types that are characterized by high biosynthetic activity and increased energy turnover. The creatine kinase system, consisting of creatine kinase isoenzymes and creatine transporter, is known to be important to support the high energy demands in such cells. We analyzed the presence and the localization of these proteins in murine and human skin under healthy and pathologic conditions, using immunoblotting and confocal immunohistochemistry with our recently developed specific antibodies. In murine skin, we found high amounts of brain-type cytosolic creatine kinase coexpressed with lower amounts of ubiquitous mitochondrial creatine kinase, both mainly localized in suprabasal layers of the epidermis, different cell types of hair follicles, sebaceous glands, and the subcutaneous panniculus carnosus muscle. With exception of sebaceous glands, these cells were also expressing creatine transporter. Muscle-type cytosolic creatine kinase and sarcomeric mitochondrial creatine kinase were restricted to panniculus carnosus. Immediately after wounding of murine skin, brain-type cytosolic creatine kinase and a creatine transporter-subspecies were transiently upregulated about 3-fold as seen in immunoblots, whereas the amount of ubiquitous mitochondrial creatine kinase increased during days 10–15 after wounding. Healthy and psoriatic human skin showed a similar coexpression pattern of brain-type cytosolic creatine kinase, ubiquitous mitochondrial creatine kinase, and creatine transporter in this pilot study, with creatine transporter species being upregulated in psoriasis. Skin comprises many cell types that are characterized by high biosynthetic activity and increased energy turnover. The creatine kinase system, consisting of creatine kinase isoenzymes and creatine transporter, is known to be important to support the high energy demands in such cells. We analyzed the presence and the localization of these proteins in murine and human skin under healthy and pathologic conditions, using immunoblotting and confocal immunohistochemistry with our recently developed specific antibodies. In murine skin, we found high amounts of brain-type cytosolic creatine kinase coexpressed with lower amounts of ubiquitous mitochondrial creatine kinase, both mainly localized in suprabasal layers of the epidermis, different cell types of hair follicles, sebaceous glands, and the subcutaneous panniculus carnosus muscle. With exception of sebaceous glands, these cells were also expressing creatine transporter. Muscle-type cytosolic creatine kinase and sarcomeric mitochondrial creatine kinase were restricted to panniculus carnosus. Immediately after wounding of murine skin, brain-type cytosolic creatine kinase and a creatine transporter-subspecies were transiently upregulated about 3-fold as seen in immunoblots, whereas the amount of ubiquitous mitochondrial creatine kinase increased during days 10–15 after wounding. Healthy and psoriatic human skin showed a similar coexpression pattern of brain-type cytosolic creatine kinase, ubiquitous mitochondrial creatine kinase, and creatine transporter in this pilot study, with creatine transporter species being upregulated in psoriasis. creatine kinase creatine transporter muscle-type and brain-type cytosolic CK ubiquitous and sarcomeric mitochondrial CK Many cells and tissues such as sperm cells, muscle, or brain use metabolically inert creatine and phosphocreatine together with isoenzymes of creatine kinase (CK) to cope efficiently with high and/or alternating adenosine triphosphate (ATP) requirements (reviewed inWallimann et al., 1992Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the "phosphocreatine circuit" for cellular energy homeostasis.Biochem J. 1992; 281: 21-40Crossref PubMed Scopus (1518) Google Scholar;Schlattner et al., 1998Schlattner U. Forstner M. Eder M. Stachowiak O. Fritz-Wolf K. Wallimann T. Functional aspects of the X-ray structure of mitochondrial creatine kinase: a molecular physiology approach.Mol Cell Biochem. 1998; 184: 125-140Crossref PubMed Google Scholar). CK catalyzes the reversible transphosphorylation between ATP and phosphocreatine. The enzyme is therefore able to stock the free energy of ATP in the form of phosphocreatine and, vice versa, to use phosphocreatine to replenish cellular ATP pools. Expression of CK isoenzymes in vertebrates is tissue and compartment specific. Two dimeric cytosolic isoenzymes, MM-CK (muscle-type) and BB-CK (brain-type) and two octameric mitochondrial isoenzymes, sMtCK (sarcomeric) and uMtCK (ubiquitous) are encoded by separate nuclear genes. Cytosolic and mitochondrial isoenzymes are usually coexpressed, with MM-CK and sMtCK found in skeletal muscle, and BB-CK and uMtCK in brain, smooth muscle, and many other organs and tissues. The interplay between cytosolic and mitochondrial CK isoenzymes, the CK/phosphocreatine circuit, provides an efficient energy buffer and energy shuttle system that plays a pivotal role in cellular energy supply and energy homeostasis (Bessman and Carpenter, 1985Bessman S.P. Carpenter C.L. The creatine-creatine phosphate energy shuttle.Annu Rev Biochem. 1985; 54: 831-862Crossref PubMed Scopus (563) Google Scholar;Wallimann et al., 1992Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the "phosphocreatine circuit" for cellular energy homeostasis.Biochem J. 1992; 281: 21-40Crossref PubMed Scopus (1518) Google Scholar;Schlattner et al., 1998Schlattner U. Forstner M. Eder M. Stachowiak O. Fritz-Wolf K. Wallimann T. Functional aspects of the X-ray structure of mitochondrial creatine kinase: a molecular physiology approach.Mol Cell Biochem. 1998; 184: 125-140Crossref PubMed Google Scholar). The creatine transporter (CRT) is an important component of the CK/phosphocreatine system, responsible for the Na+/CL–dependent creatine uptake into the cell (Guimbal and Kilimann, 1993Guimbal C. Kilimann M.W. A Na(+)-dependent creatine transporter in rabbit brain, muscle, heart, and kidney. cDNA cloning and functional expression.J Biol Chem. 1993; 268: 8418-8421Abstract Full Text PDF PubMed Google Scholar;Sora et al., 1994Sora I. Richman J. 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This may be due to manifold energy requirements of these cells, including high proliferation rates, active ion pumps, and transport processes. Where examined, a coexpression of BB-CK and uMtCK was observed (Keller and Gordon, 1991Keller T.C. Gordon P.V. Subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells.Cell Motil Cytoskeleton. 1991; 19: 169-179Crossref PubMed Scopus (17) Google Scholar), with BB-CK being the predominant isoenzyme, by far; however, the abundance of CK in epithelia varies considerably. Certain organs contain epithelia that are highly enriched in CK, e.g., the electric organ of Torpedo (Barrantes et al., 1983Barrantes F.J. Mieskes G. Wallimann T. Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes.Proc Natl Acad Sci USA. 1983; 80: 5440-5444Crossref PubMed Scopus (23) Google Scholar), where also CRT was detected (Guimbal and Kilimann, 1994Guimbal C. Kilimann M.W. A creatine transporter cDNA from Torpedo illustrates structure/function relationships in the GABA/noradrenaline transporter family.J Mol Biol. 1994; 241: 317-324https://doi.org/10.1006/jmbi.1994.1507Crossref PubMed Scopus (35) Google Scholar), the choroid plexus (Kaldis et al., 1996Kaldis P. Hemmer W. Zanolla E. Holtzman D. Wallimann T. "Hot spots" of creatine kinase localization in brain: cerebellum, hippocampus and choroid plexus.Dev Neurosci. 1996; 18: 542-554Crossref PubMed Scopus (72) Google Scholar), kidney (Ikeda, 1988Ikeda K. Localization of brain type creatine kinase in kidney epithelial cell subpopulations in rat.Experientia. 1988; 44: 734-735Crossref PubMed Scopus (10) Google Scholar), the adult eye lens (Friedman et al., 1989Friedman D.L. Hejtmancik J.F. Hope J.N. Perryman M.B. Developmental expression of creatine kinase isozymes in mammalian lens.Exp Eye Res. 1989; 49: 445-457Crossref PubMed Scopus (12) Google Scholar), or prostate (Silverman et al., 1979Silverman L.M. Dermer G.B. Zweig M.H. Van Steirteghem A.C. Tokes Z.A. Creatine kinase BB. a new tumor-associated marker.Clin Chem. 1979; 25: 1432-1435Crossref PubMed Scopus (80) Google Scholar). Lower amounts of BB-CK were found in many epithelia of the urogenital system and the digestive tract (Wold et al., 1981Wold L.E. Li C.Y. Homburger H.A. Localization of the B and M polypeptide subunits of creatine kinase in normal and neoplastic human tissues by an immunoperoxidase technic.Am J Clin Pathol. 1981; 75: 327-332PubMed Google Scholar;Gordon and Keller, 1992Gordon P.V. Keller T.C. Functional coupling to brush border creatine kinase imparts a selective energetic advantage to contractile ring myosin in intestinal epithelial cells.Cell Motil Cytoskeleton. 1992; 21: 38-44Crossref PubMed Scopus (11) Google Scholar;Sistermans et al., 1995Sistermans E.A. De Kok Y.J. Peters W. Ginsel L.A. Jap P.H. Wieringa B. Tissue- and cell-specific distribution of creatine kinase B. a new and highly specific monoclonal antibody for use in immunohistochemistry.Cell Tissue Res. 1995; 280: 435-446Crossref PubMed Scopus (56) Google Scholar). In epithelia of the respiratory tract, only traces of CK were observed (Braegger, Schlattner, Utiger, Wallimann, and Sennhauser, unpublished data). Although representing one of the largest epithelia, skin has not yet been analyzed in detail for CK isoenzymes and their tissue localization. Furthermore, no information at all has been published on the presence and localization of CRT in this tissue. CK activity was reported in extracts of normal and psoriatic human skin, skin tumors, and keratinocyte cell cultures (Zemtsov et al., 1994aZemtsov A. Cameron G.S. Bradley C.A. Montalvo-Lugo V. Mattioli F. Identification and activity of cytosol creatine phosphokinase enzymes in normal and diseased skin.Am J Med Sci. 1994; 308: 365-369Crossref PubMed Scopus (6) Google Scholar) and BB-CK was localized in human skin epidermis and blister fluid (Kiistala et al., 1989Kiistala U. Paavonen T. Saarelainen I. et al.Epidermis is the origin of high creatine kinase levels in skin blister fluid.Acta Derm Venereol. 1989; 69: 284-287PubMed Google Scholar), as well as in murine skin (Chida et al., 1990Chida K. Kasahara K. Tsunenaga M. Kohno Y. Yamada S. Ohmi S. Kuroki T. Purification and identification of creatine phosphokinase B as a substrate of protein kinase C in mouse skin in vivo.Biochem Biophys Res Commun. 1990; 173: 351-357Crossref PubMed Scopus (32) Google Scholar;Sistermans et al., 1995Sistermans E.A. De Kok Y.J. Peters W. Ginsel L.A. Jap P.H. Wieringa B. Tissue- and cell-specific distribution of creatine kinase B. a new and highly specific monoclonal antibody for use in immunohistochemistry.Cell Tissue Res. 1995; 280: 435-446Crossref PubMed Scopus (56) Google Scholar). Phosphocreatine in skin has been detected by high-performance liquid chromatography (Zemtsov et al., 1993Zemtsov A. Cameron G.S. Stadig B. Martin J. Measurement of phosphocreatine in cutaneous tissue by high pressure liquid chromatography.Am J Med Sci. 1993; 305: 8-11Crossref PubMed Scopus (16) Google Scholar) and more unambiguously by high-resolution 31P-NMR imaging (Bohning et al., 1996Bohning D.E. Wright A.C. Spicer K.M. In vivo phosphorous spectroscopy of human skin.Magn Reson Med. 1996; 35: 186-193Crossref PubMed Scopus (11) Google Scholar), indicating a functional phosphocreatine/CK system in this tissue. In addition to basic cellular functions of skin, the CK/phosphocreatine system may also be involved in repair processes like wound healing or in hyperproliferative skin disease, such as psoriasis. During wound healing, enzymes of the de novo nucleotide biosynthetic pathway (Gassmann et al., 1999Gassmann M.G. Stanzel A. Werner S. Growth factor-regulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor action.Oncogene. 1999; 18: 6667-6676Crossref PubMed Scopus (22) Google Scholar) are highly upregulated in the wounded epidermis, reflecting the elevated need for nucleoside triphosphates in cellular energy transduction, nucleic acid synthesis, and biosynthetic pathways in general. Similar conclusions may be true for psoriasis, where highly elevated phosphocreatine levels point to an especially active CK/phosphocreatine system (Zemtsov et al., 1994bZemtsov A. Dixon L. Cameron G. Human in vivo phosphorous 31 magnetic resonance spectroscopy of psoriasis.J Am Acad Dermatol. 1994; 30: 959-965Abstract Full Text PDF PubMed Scopus (14) Google Scholar). Psoriasis is an inflammatory skin disease that is characterized by increased angiogenesis and hyperthickening of the epidermis due to an imbalance in keratinocyte proliferation and differentiation (Kadunce and Krueger, 1995Kadunce D.P. Krueger G.G. Pathogenesis of psoriasis.Dermatol Clin. 1995; 13: 723-737PubMed Google Scholar). In this study, we set out to identify and localize CK and CRT species in healthy and diseased skin, as well as during the wound healing process. Using highly specific antibodies for immunoblotting and immunohistochemistry with fluorescence and confocal microscopy, we could show coexpression of BB-CK, uMtCK, and CRT in several specific cell types and changes in protein levels during wound healing and in psoriatic skin disease. CK isoenzymes were expressed in Escherichia coli and purified as described earlier (Eder et al., 1999Eder M. Schlattner U. Becker A. Wallimann T. Kabsch W. Fritz-Wolf K. Crystal structure of brain type creatine kinase at 1.41 Å resolution.Prot Sci. 1999; 8: 2258-2269Crossref PubMed Scopus (97) Google Scholar;Schlattner et al., 2000Schlattner U. Eder M. Dolder M. Khuchua Z.A. Strauss A.W. Wallimann T. Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes.Biol Chem. 2000; 381: 1063-1070Crossref PubMed Scopus (44) Google Scholar). Final gel filtration chromatography on Superose 12 (Amersham Pharmacia Biotech, Uppsala, Sweden) was used to obtain highly pure protein. Polyclonal antibodies against human sMtCK and uMtCK were produced in rabbits by standard procedures. Rabbits were injected subcutaneously with 200 µg aliquots of protein in complete Freund's adjuvant, followed by three boosts, every 3 wk, in incomplete Freund's adjuvant. Immune sera were collected 3, 5, and 7 wk after the last immunization. Polyclonal antibodies against human BB-CK were obtained from egg yolk of immunized SHAVER outdoor 579 hens. Forty micrograms of BB-CK in complete Freund's adjuvant were injected into the pectoral muscle of each hen, followed after 8 wk by one boost of 40 μg BB-CK in incomplete Freund's adjuvant. Eggs were collected after one further week and stored at 4°C. A simple water dilution method was used to separate yolk plasma proteins from the granules and lipids (Akita and Nakai, 1993Akita E.M. Nakai S. Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.J Immunol Methods. 1993; 160: 207-214Crossref PubMed Scopus (232) Google Scholar;Kokko and Kärenlampi, 1998Kokko H.K.I. Kärenlampi S. Rapid production of antibodies in chicken and isolation from eggs.in: Celis J.E. Cell Biology, A Laboratory Handbook. Academic Press, San Diego1998: 410-417Google Scholar). The egg yolk was separated, washed and diluted with 9 vol. of distilled water. The emulsion was adjusted to pH 5.0, incubated overnight at 4°C, and centrifuged at 8000 × g for 25 min at 4°C. Proteins containing egg immunoglobulins (IgY) were precipitated with sodium acetate (200 g per l) at room temperature and resuspended in 5–8 ml Tris-buffered saline (TBS) per egg. BB-CK-specific IgY were further purified by affinity chromatography, using human BB-CK coupled to N-hydroxysuccinimide-ester activated methacrylate copolymer (Affi-Prep 10, Bio-Rad, Reinach, Switzerland). Bound IgY was eluted by 0.2 m glycine, 0.15 m NaCl, pH 2.3, neutralized in 1 m Tris–HCl buffer, pH 8.0, and concentrated by ultrafiltration (Centriplus YM-100, Millipore, Bedford, MA). For extended storage, IgY preparations were adjusted to 40% glycerol and stored at - 20°C. Purity and recovery of IgY were monitored by 8% nonreducing standard sodium dodecyl sulfate (SDS)–polyacrylamide gels and Coomassie staining. BALB/C mice were obtained from RCC (Füllinsdorf, Switzerland). They were housed and fed according to Swiss federal guidelines, and all procedures were approved by the local authorities. Full thickness excisional wounds were generated as described earlier (Werner et al., 1994Werner S. Smola H. Liao X. Longaker M.T. Krieg T. Hofschneider P.H. Williams L.T. The function of KGF in morphogenesis of epithelium and reepithelialization of wounds.Science. 1994; 266: 819-822Crossref PubMed Scopus (488) Google Scholar). Briefly, mice (8–12 wk of age) were anesthetized with a single intraperitoneal injection of ketamine/xylazine. The hair on the animals' back was shaved and the skin was wiped with 70% ethanol. Four full-thickness excisional wounds (5 mm diameter, 3–4 mm apart, two wounds on each side of the spinal cord) were generated on the back of each animal by excising skin and panniculus carnosus. The wounds were allowed to dry to form a scab. The back skin that was excised upon generation of the wounds served as control (0 d, healthy skin). Animals were killed at different time points after injury (1–14 d). For tissue extracts, the wounds on the left side, including 2 mm of the epithelial margins were isolated and immediately frozen in liquid nitrogen. Wound tissue of several animals was pooled and soluble proteins were extracted in lysis buffer containing NP-40 and urea as described elsewhere (Werner et al., 1993Werner S. Weinberg W. Liao X. et al.Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.EMBO J. 1993; 12: 2635-2643Crossref PubMed Scopus (214) Google Scholar;Wakita and Takigawa, 1999Wakita H. Takigawa M. Activation of epidermal growth factor receptor promotes late terminal differentiation of cell-matrix-disrupted keratinocytes.J Biol Chem. 1999; 274: 37285-37291Crossref PubMed Scopus (52) Google Scholar). For immunohistochemistry, the complete wounds on the right side were isolated, bisected, fixed in 95% ethanol/1% acetic acid, and embedded in paraffin. Human tissue samples (brain, kidney, heart, and skeletal muscle) were obtained from Dr V. Adams (University of Leipzig, Germany), and soluble protein and mitochondria-enriched pellets were extracted according toSchlegel et al., 1988Schlegel J. Wyss M. Schurch U. et al.Mitochondrial creatine kinase from cardiac muscle and brain are two distinct isoenzymes but both form octameric molecules.J Biol Chem. 1988; 263: 16963-16969Abstract Full Text PDF PubMed Google Scholar. Human skin biopsies of three healthy and three psoriasis patients containing epidermis and dermis of upper or lower extremities were obtained from Dr. C. Mauch (University of Cologne, Germany; seeHanselmann et al., 2001Hanselmann C. Mauch C. Werner S. Haem oxygenase-1. a novel player in cutaneous wound repair and psoriasis.Biochem J. 2001; 353: 459-466https://doi.org/10.1042/0264-6021:3530459Crossref PubMed Scopus (111) Google Scholar). They were immediately frozen without fixing procedure. Protein extracts were prepared as described previously (Werner et al., 1993Werner S. Weinberg W. Liao X. et al.Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.EMBO J. 1993; 12: 2635-2643Crossref PubMed Scopus (214) Google Scholar). Samples were separated by standard 12% SDS–polyacrylamide gel electrophoresis and electrotransferred by semidry blotting (Trans Blot SD, Bio-Rad) on to nitrocellulose (0.45 μm pore size; Schleicher & Schüll, Dassel, Germany) according to the manufacturer's instructions. Loading of the blots was verified by a reversible staining with Ponceau S (0.2% in 0.3% trichloroacetic acid (TCA); Serva, Heidelberg, Germany). Membranes were blocked with 4% fat-free milk powder in TBS (20 mm Tris–HCl, pH 7.5, 150 mm NaCl), incubated for 1 h with CK or CRT immune sera (see below, 1:1000 dilution in blocking buffer) or affinity-purified purified anti-BB-CK IgY (1:500 dilution in blocking buffer), washed three times in TBS, incubated 1 h with peroxidase-coupled secondary antibodies (1:3000 dilution in blocking buffer), either goat anti-rabbit IgG (Nordic, Lausanne, Switzerland) or rabbit anti-chicken IgY (Jackson ImmunoResearch, West Grove, PA) and finally washed three times with TBS. Blots were developed with enhanced chemiluminescence substrate (NEN, Zaventem, Belgium) and exposed to X-ray film (1–60 s) or analyzed with a digital imager (Kodak Image Station; Kodak, Rochester, NY). Ethanol/acetic-acid fixed and paraffin wax embedded samples of mouse skin and nonfixed samples of human skin were cut with a microtome into 5–7 μm thin sections. Paraffin-embedded sections were deparaffinized, rehydrated, and incubated in 0.05% sodium borohydride (NaBH4) in phosphate-buffered saline (PBS) to quench autofluorescence. After rinsing with PBS, sections were permeabilized with 0.2% Triton-X-100 for 10 min, followed by a wash in PBS. Frozen sections stored at -80°C were directly fixed in acetone at -20°C for 10 min, washed three times in PBS, and blocked for 30 min with 5% goat serum albumin/1% bovine serum albumin (BSA). Sections were then incubated overnight at 4°C in a moist chamber with different primary antibodies, diluted in PBS, containing either 0.2% Tween-20 (polyoxyethylenesorbitan monolaureate) and 1% BSA (paraffin-embedded) or 5% goat serum albumin/1% BSA (frozen sections). Polyclonal primary antibodies were (dilution in brackets): affinity-purified chicken IgY against human BB-CK (lot 23–1; 1:100), preimmune IgY (1:100), and rabbit anti-sera against human sMtCK (lot r3i3; 1:200), human uMtCK (lot r7i3; 1:200), chicken MM-CK (lot 1985; 1:100), and rat CRT C-terminus (lot 75898; 1:100), as well as the corresponding rabbit preimmune sera (same dilutions). Monoclonal primary antibodies were (dilution in brackets): anti-keratin 10 and keratin 14 (Dako Diagnostics AG, Zug, Switzerland; 1:100); anti-CD31 (Dako; prediluted), anti-F4/80 (Serotec, Oxford, GB; 1:100), and anti-wheat germ agglutinin (Dako; 1:200). After washing four times in PBS/0.1% Tween-20, sections were incubated for 1 h at room temperature in a moist chamber with secondary antibody diluted in PBS, containing either 0.1% Tween-20 and 12% BSA (paraffin embedded) or only 1% BSA (frozen sections). Secondary antibodies were Cy3-conjugated rabbit or donkey anti-chicken IgY (Jackson ImmunoResearch; 1:500), Texas Red-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch; 1:100), Cy3- or fluorescein isothiocyanate-conjugated goat anti-rabbit or anti-mouse IgG (Pierce, Rockford, IL; 1:200–1:500). Sections were again washed four times in PBS, including 0.1% Tween-20 and 0.02% sodium azide, and finally mounted in an anti-fading solution (30 mm Tris-HCl, pH 9.5, 70% (vol/vol) glycerol, and 240 mm N-propyl-gallate). Fluorescence images of sections were recorded on a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) with an attached CCD camera (Hamamatsu 3CCD, Hamamatsu, Herrsching, Germany) using Achrostigmat 5×/0.12 or Plan-Neofluoar 10×/0.3 objectives. Confocal images were recorded with a Leica inverted microscope DM IRB/E connected to a Leica true confocal scanner TCS NT and Leica PL APO 100×/1.4 oil or PL APO 63×/1.4 oil immersion objectives. The system was equipped with an argon/krypton mixed gas laser. Image processing was done on a Silicon Graphics workstation using Imaris software (Bitplane AG, Zurich, Switzerland) and Corel Photo-Paint (Corel, Ottawa, Canada). For immunohistochemical studies, we have raised polyclonal antibodies against heterologously expressed and highly purified human CK isoenzymes in rabbit and chicken. Rabbit anti-sera against human sMtCK and uMtCK specifically detected mitochondrial isoenzymes, with relatively high selectivity for the corresponding isoenzyme. Anti-uMtCK anti-sera (Figure 1a) visualized human uMtCK in mitochondria-enriched brain extracts (Figure 1a, lane 2) and purified uMtCK (Figure 1a, lane 5), with some cross-reactivity with abundant sMtCK in muscle (Figure 1a, lane 4) and pure sMtCK (Figure 1a, lane 6). Anti-sMtCK anti-sera (Figure 1b) reacted mainly with human sMtCK in mitochondria-enriched muscle extracts (Figure 1b, lane 4) or purified sMtCK (Figure 1b, lane 6), with minor cross-reaction with pure uMtCK (Figure 1b, lane 5). Antibodies did neither cross-react with other proteins in several human tissue extracts nor with cytosolic CK (Figure 1, lanes 1–4), except for a faint cross-reactivity of sMtCK antibodies with cytosolic MM-CK in human muscle extracts, running just below the sMtCK band (Figure 1b, lane 3); however, no signal was observed with chicken MM-CK (Figure 1b, lane 8). Chicken IgY against heterologously expressed and purified human BB-CK were isolated from egg yolk and affinity-purified to eliminate any cross-reactivity with other proteins (Figure 2a). The final IgY preparation only reacted with tissue containing BB-CK, such as brain (Figure 2b, lane 1) and kidney (Figure 2b, lane 2), as well as with purified human BB-CK (Figure 2b, lanes 7, 8). Rabbit polyclonal antibodies against the C-terminal part of rat CRT have already been described elsewhere (Guerrero-Ontiveros and Wallimann, 1998Guerrero-Ontiveros M.L. Wallimann T. Creatine supplementation in health and disease. Effects of chronic creatine ingestion in vivo: down-regulation of the expression of creatine transporter isoforms in skeletal muscle.Mol Cell Biochem. 1998; 184: 427-437Crossref PubMed Google Scholar). All antibodies reacted in a similar way with CK and CRT from rat and mouse tissue extracts (data not shown). Sections of paraffin-embedded murine skin samples were analyzed by immunohistochemistry for the presence of different CK isoenzymes and CRT (Figure 3, left panel). Fluorescence microscopy revealed strong and cell-type specific expression of BB-CK, uMtCK, and CRT (Figure 3c, f, l) in epidermis, hair follicles, and to a lesser extent in panniculus carnosus, the subcutaneous striated muscle. Dermis and subcutis (adipose tissue) stained much weaker for BB-CK, uMtCK, and CRT. Signals for sMtCK (Figure 3i) and MM-CK (data not shown) were restricted to panniculus carnosus and therefore not due to cross-reactivity with uMtCK or BB-CK. In 3 d old murine skin wounds, the hyperproliferative epithelium at the wound edge stained especially strong for BB-CK, uMtCK, and CRT (Figure 3, central panel). Control incubation with preimmune IgY and preimmune rabbit sera (Figure 3, right panel) showed only very faint background staining, confirming the low cross-reactivity of the applied antibodies in mouse tissue. Confocal microscopy of murine skin sections revealed expression of BB-CK in specific cell types (Figure 4, left panels). In murine epidermis, the suprabasal layers costained with keratin 10 (Figure 4b) rather than the basal layers costained with keratin 14 (Figure 4a) were strongly positive for BB-CK. As evident in Figure 4b, BB-CK was mainly located in the cell periphery near or at the plasma membrane, showing a particulate pattern. In the dermis, the majority of cells in the pilosebaceous apparatus were positive for BB-CK (Figure 4e, f, i). The enzyme was especially abundant in the sebaceous glands and specific parts of the hair follicle, including the dermal hair papilla (Figure 4e) and the inner root sheath, which is the cell layer surrounding the growing hair (Figure 4e, f, i). By contrast, the outer root sheath costained with keratin 14 is nearly devoid of BB-CK (Figure 4f). Strong staining was also detectable in endothelial cells of blood vessels in the subcutis and in the panniculus carnosus (Figure 4j). Among the CK-positive cells in the granulation tissue of wounds we identified endothelial cells and macrophages, using costaining with CD-31 and F4/80, respectively (data not shown;Austyn and Gordon, 1981Austyn J.M. Gordon S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage.Eur J Immunol. 1981; 11: 805-815Crossref PubMed Scopus (1239) Google Scholar). Staining for uMtCK in all these sections was nearly identical to BB-CK (data not shown, compare to Figure 3f, g). Most cells strongly expressi
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