Pantothenic acid is one of the essential nutrients contained in various foods. Stress, pregnancy, and predominant consumption of processed foods can cause deficiency.
Pantothenic acid is one of the essential nutrients contained in various foods. Stress, pregnancy, and predominant consumption of processed foods can cause deficiency.
Pantothenic acid is one of the essential nutrients contained in various foods. Stress, pregnancy, and predominant consumption of processed foods can cause deficiency.
The Japanese Pharmacological Society J Pharmacol Sci 115, 230 234 (2011) Pantothenic acid is one of the essential nutrients con- tained in various foods. Pantothenic acid, also referred to as vitamin B5, is a member of the water-soluble group of vitamins. Pantothenic acid is converted to 4-phospho- pantetein, which is then converted to coenzyme A (CoA) with ATP. CoA is involved in acyl transfer during me- tabolism of carbohydrates, amino acids, and lipids. CoA is also utilized for synthesis of acyl carrier protein (ACP), which is mainly involved in lipid metabolism. Although pantothenic acid deficiency is rare in healthy subjects, factors such as stress, pregnancy, and predominant con- sumption of processed foods can cause pantothenic acid deficiency. It has been reported that pantothenic acid deficiency causes growth arrest, weight loss, dermatitis, dyslipidemia, neuropathy, and adrenal disorders (1, 2). Pantothenic acid and its alcohol analogue panthenol have been used for many years for skin protection and to improve skin regeneration after wounding (3). In terms of the effects of pantothenic acid on dermal fibroblasts, Weimann and Hermann (4) revealed that Ca D-panto- thenic acid promoted fibroblast proliferation and migra- tion. Furthermore, Wiederholt et al. (5) used microarray analysis to investigate the effects of Ca D-pantothenic acid on gene expression in proliferating dermal fibro- blasts. However, there are no studies examining the effect of pantothenic acid on keratinocytes. Therefore, in this study, we examined the direct effect of pantothenic acid on the proliferation and differentiation of keratinocytes by using the MTT assay and measuring the expression of cytokeratin 10 (CK10), a keratinocyte differentiation marker (6). We also evaluated the effect of pantothenic acid deficiency on the synthesis of keratinocyte growth factor (KGF) and procollagen in fibroblasts, which are involved in keratinocyte growth. Human HaCaT keratinocytes were given to us by Dr. N.E. Fusenig (German Cancer Research Center, Heidelberg, Germany). The cells were grown in Dul- beccos modified Eagles medium (DMEM) containing 4 mg/l Ca D-pantothenic acid supplemented with 1% fetal calf serum (FCS), penicillin (50 units/ml), and strepto- mycin (50 g/ml) in a humidified atmosphere of 5% CO 2 / 95% air at 37C. Keratinocytes were treated with pantothenic aciddeficient DMEM for a week before the experiments. Fibroblasts were prepared as follows: Em- bryonic day 15.5 mice were dissected and the whole body except head and extremities were homogenized, *Corresponding author. nakahata@mail.pharm.tohoku.ac.jp Published online in J-STAGE on January 18, 2011 (in advance) doi: 10.1254/jphs.10224SC The Effect of Pantothenic Acid Deficiency on Keratinocyte Proliferation and the Synthesis of Keratinocyte Growth Factor and Collagen in Fibroblasts Daisaku Kobayashi 1 , Miho Kusama 1 , Masaaki Onda 2 , and Norimichi Nakahata 1, * 1 Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan 2 Daiichi Fine Chemical Co., Ltd., 530 Chokeiji, Takaoka, Toyama 933-8511, Japan Received August 27, 2010; Accepted December 3, 2010 Abstract. It has been reported that pantothenic acid (vitamin B5) and panthenol, an alcohol de- rivative of pantothenic acid, have beneficial moisturizing effects on the skin. However, few studies have investigated the mechanism of action of pantothenic acid on skin tissues. We tried to clarify the role of pantothenic acid on skin function by using keratinocytes and fibroblasts. The depletion of pantothenic acid from the culture medium suppressed keratinocyte proliferation and promoted differentiation. Moreover, pantothenic acid depletion decreased the synthesis of keratinocyte growth factor and procollagen 4a2 in fibroblasts. These results suggest that pantothenic acid is essential for maintaining keratinocyte proliferation and differentiation. Keywords: collagen, keratinocyte, pantothenic acid Short Communication 231 Pantothenic Acid Regulates Skin Functions washed with phosphate-buffered saline (PBS) many times. They were cultured throughout in normal DMEM or pantothenic aciddeficient DMEM supplemented with 10% FCS, penicillin (50 units/ml), and streptomycin (50 g/ml) in a humidified atmosphere of 5% CO 2 / 95% air at 37C for a week and used for the experiments. Cell viability was measured by the previously described MTT assay (7). The extraction of total RNA and the prepara- tion of first-strand cDNA was done as previously de- scribed (8). The real-time PCR conditions were as fol- lows: 94C for 10 s, followed by several cycles of 10 s at 94C, 20 s at the annealing temperature shown in Table 1, and 30 s at 72C. The mRNA expressions of CK10, KGF, and procollagen 1a1, 1a2, 4a1, and 4a2 were each normalized by the expression of -actin. The protein synthesis of CK10 and procollagen 4a2 were analyzed by SDS-polyacrylamide gel electrophoresis and immunob- lotting. HaCaT cells and mouse fibroblasts were dis- solved in Laemmli sample buffer (final concentration: 75 mM Tris-HCl, 2% SDS, 15% glycerol, and 3% 2-mer- captoethanol, pH 6.8) and heated at 95C for 5 min. Electrophoresis was performed on 10% or 8% acrylamide gels. Proteins were transferred electrically from the gel onto a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA) by a semidry blotting method. The blots were blocked for 1 h with 5% skim milk in tris-buffered saline supplemented with 0.1% Tween 20, and then they were incubated with a 1:1000 dilution mouse anti-CK10 antibody (Thermo Fisher Scientific, Inc., Waltham, MA, USA) or rabbit anti-pro- collagen 4a2 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4C. After several washes, the blots were incubated at 25C for 1 h with a 1:4000 dilution of secondary antibody (horseradish per- oxidaseconjugated anti-rabbit IgG antibody or anti- mouse IgG antibody). Blots were developed using an enhanced chemiluminescence on Hyperfilm. Data are expressed as means S.E.M. Significant differences were determined by Students t-test or Mann-Whitneys U test. It has been shown that pantothenic acid affects cell proliferation of dermal fibroblasts (4). However, there are no studies investigating the effect of pantothenic acid on keratinocyte proliferation. Therefore, we first investi- gated the effect of pantothenic acid deficiency on the proliferation of HaCaT keratinocytes using the MTT as- say (Fig. 1A) and cell counting (Fig. 1B). The MTT assay and cell counting revealed that the proliferation of HaCaT keratinocytes cultured in pantothenic aciddeficient medium was suppressed compared with HaCaT keratino- cytes cultured in the control medium. Then, we examined the effect of pantothenic aciddeficient medium on the differentiation of keratinocytes, determining the express- tion of CK10, a keratinocyte differentiation marker (6). The mRNA expression of CK10 (Fig. 1C) and the protein level of CK10 (Fig. 1D) were promoted in pantothenic aciddeficient medium. These data suggest that the dif- ferentiation of HaCaT keratinocytes cultured in panto- thenic aciddeficient medium was enhanced compared with HaCaT keratinocytes cultured in the control medium. The low proliferative ability and enhanced differentiation of keratinocytes cultured in pantothenic aciddeficient DMEM indicate that the depletion of pantothenic acid may decelerate or arrest the cell cycle, thus enhancing keratinocyte differentiation (9). It is known that the bal- ance between proliferation and differentiation is regulated Table 1. Primers for reverse transcription-polymerase chain reaction used in the present study Sequence Accession number Annealing temperature (C) CK10 KGF Procollagen 1a1 Procollagen 1a2 Procollagen 4a1 Procollagen 4a2 -Actin Sense: Anti-sense: Sense: Anti-sense: Sense: Anti-sense: Sense: Anti-sense: Sense: Anti-sense: Sense: Anti-sense: Sense: Anti-sense: 5-GGATGAGCTGACCCTGACCAA-3 5-GCAGCATTCATTTCCACATTCAC-3 5-TTTGGAAAGAGCGACGACTT-3 5-GGCAGGATCCGTGTCAGTAT-3 5-GCAACATGGAGACAGGTCAG-3 5-TCGCTTCCGTACTCGAACG-3 5-CCGTTGGCAAAGATGGTAG-3 5-CGTTGTCGTAGCAGGGTTCT-3 5-TTCGTTGGCCTCTGTTTGC-3 5-TTGGGGCTGAAACGATGG-3 5-CCAGCACTCATTCCAACC-3 5-GACTGCAGGAAGGTGAGC-3 5-AGGGAAATCGTGCGTGACAT-3 5-TCCTGCTTGCTGATCCACAT-3 NM_000421.3 NM_008008.4 NM_007742.3 NM_007743.2 NM_009931.2 NM_009932.3 NM_03114 60 56 56 56 56 56 56 232 D Kobayashi et al by various factors, and protein kinase C or tyrosine kinase may be involved in the switch from proliferation to differentiation (10), although the detailed mechanism has not been reported. Moreover, it has been shown that an increased intracellular Ca 2+ concentration or high cell density might induce cell differentiation (11). Our data suggest that pantothenic aciddeficiency might induce keratinocyte differentiation directly, accompanied with growth arrest, although the mechanism of the pantothenic acid is still unknown. Fibroblasts secrete various kinds of growth factors and active substrates, which regulate keratinocyte prolifera- tion and differentiation (12). Then, we examined the synthesis of KGF, also referred to FGF7, and procollagen in fibroblasts cultured in pantothenic aciddeficient medium. It is known that KGF is secreted by fibroblasts and promotes keratinocyte proliferation and migration (12). Our results indicated that the mRNA expression of KGF was decreased in pantothenic aciddeficient fibroblasts (Fig. 2A). Since the suppression of the KGF level causes the growth arrest of keratinocytes, it is possible that pantothenic acid promotes the growth of keratinocytes via the synthesis of KGF. Collagen is categorized into more than twenty func- tional types, including fibrous collagen (type I, II, III, V, XI), basement membrane collagen (type IV), long-chain collagen (type VII), and short-chain collagen (VIII, X). All types of collagen are important for the regulation of the function of epidermal cells (13). Next we examined the synthesis of collagens in fibloblasts. First, we focused on type I collagen and evaluated procollagen 1a1 and 1a2 mRNA expression. Procollagen is a precursor of mature collagen, and type I collagen consists of two procollagen 1a1 and one procollagen 1a2 fibers. We found that there was no difference in type I procollagen mRNA expres- sion between the normal medium and the pantothenic aciddeficient medium (Fig. 2: B, C). Next, the expres- sion of type IV basement membrane procollagen was examined. We found that the mRNA expression of pro- collagen 4a1 and 4a2 was partially suppressed in fibro- blasts cultured in the pantothenic aciddeficient medium (Fig 2: D, E). Furthermore, the protein level of procol- lagen 4a2 was also decreased in fibroblasts cultured in the pantothenic aciddeficient medium (Fig. 2F). Thus, pantothenic acid may be involved in the biosynthesis of type IV collagen, which is important for the adherence and growth of epidermal cells (14). (A) Control PA (-) A b s o r b a n c e
( 5 9 5
n m ) 0 0.5 1.0 1.5 2.0 * Control PA (-) 0 0.2 0.4 0.6 0.8 1.0 1.2 C e l l
n u m b e r ( F o l d
o f
c o n t r o l ) * (B) (C) Control PA (-) 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 * C K 1 0
m R N A
e x p r e s s i o n ( F o l d
o f
c o n t r o l ) (D) Control -Actin Cytokeratin 10 PA (-) Fig. 1. Effect of pantothenic acid deficiency on the proliferation of HaCaT keratinocytes. Keratinocytes were cultured in normal DMEM or pantothenic aciddeficient DMEM. A) Cell growth was measured by the MTT assay. Data are expressed as means S.E.M. of three determinations. *P < 0.05 vs. control. B) The cells were detached and the cell number was counted. Data are expressed as means S.E.M. of 11 determinations. *P < 0.05 vs. control. C) CK10 mRNA expression was analyzed by RT- PCR. Data are expressed as means S.E.M. of six determinations. *P < 0.05 vs. control. D) CK10 protein expression was detected by Western blotting. 233 Pantothenic Acid Regulates Skin Functions Keratinocyte growth and differentiation are essential for epidermal barrier function (15). The present results suggest that pantothenic acid may regulate epidermal barrier function through the proliferation and differentia- tion of keratinocytes directly or indirectly via the synthe- sis of KGF and type IV collagen. Acknowledgments This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (No, 19659011 and 20054002 to N.N.) and by a grant from the Smoking Research Foundation (N.N.). This work was also partly supported by the Sasakawa Scientific Research Grant from The Japan Science Society (D.K.). References 1 Olson RE, Kaplan NO. The effect of pantothenic acid deficiency upon the coenzyme A content and pyruvate utilization of rat and duck tissues. J Biol Chem. 1948;175:515529. 2 Eisenstein AB. Pantothenic acid and adrenocortical hormone se- cretion. Endocrinology. 1957;60:298302. 3 Ebner F, Heller A, Rippke F, Tausch I. Topical use of dexpanthe- nol in skin disorders. Am J Clin Dermatol. 2002;3:427433. 4 Weimann BI, Hermann D. Studies on wound healing: effects of Control PA (-) 0 0.2 0.4 0.6 0.8 1.0 1.2 * K G F
m R N A
e x p r e s s i o n ( F o l d
o f
c o n t r o l ) Control PA (-) 0 0.2 0.4 0.6 0.8 1.0 1.2 N.S. P r o c o l l a g e n
1 a 1
m R N A ( F o l d
o f
c o n t r o l ) (A) (B) (C) Control PA (-) 0 0.2 0.4 0.6 0.8 1.0 1.2 N.S. P r o c o l l a g e n
1 a 2
m R N A ( F o l d
o f
c o n t r o l ) (D) Control PA (-) 0 0.2 0.4 0.6 0.8 1.0 1.2 * P r o c o l l a g e n
4 a 1
m R N A ( F o l d
o f
c o n t r o l ) (E) Control PA (-) 0 0.2 0.4 0.6 0.8 1.0 1.2 P r o c o l l a g e n
4 a 2
m R N A ( F o l d
o f
c o n t r o l ) * (F) -Actin Procollagen 4a2 Control PA (-) Fig. 2. Effect of pantothenic acid deficiency on the synthesis of KGF and procollagen in mouse fibroblasts. Fibroblasts were cultured in normal DMEM or pantothenic aciddeficient DMEM. A) KGF mRNA expression was analyzed by RT-PCR. Data are expressed as means S.E.M. of three determinations. *P < 0.05 vs. control. B, C) Procollagen 1a1 (B) and 1a2 (C) mRNA ex- pression was analyzed by RT-PCR. Data are expressed as means S.E.M. of three determinations. D, E) Procollagen 4a1 (D) and 4a2 (E) mRNA expressions were analyzed by RT-PCR. Data are expressed as means S.E.M. of four determinations. *P < 0.05 vs. control. F) Procollagen 4a2 protein expression was analyzed by Western blotting. 234 D Kobayashi et al calcium D-pantothenate on the migration, proliferation and pro- tein synthesis of human dermal fibroblasts in culture. Int J Vitam Nutr Res. 1999;69:113119. 5 Wiederholt T, Heise R, Skazik C, Marquardt Y, Joussen S, Erdmann K, et al. Calcium pantothenate modulates gene expres- sion in proliferating human dermal fibroblasts. Exp Dermatol. 2009;18:969978. 6 Matus CE, Ehrenfeld P, Pavicic F, Sarmiento JM, Astroza A, Sanchez T, et al. Activation of kinin B receptor triggers differen- tiation of cultured human keratinocytes. Br J Dermatol. 2008; 159:792803. 7 Ohkubo S, Nagata K, Nakahata N. Adenosine uptake-dependent C6 cell growth inhibition. Eur J Pharmacol. 2007;577:3543. 8 Kobayashi D, Ohkubo S, Nakahata N. Cooperation of calcineurin and ERK for UTP-induced IL-6 production in HaCaT keratino- cytes. Eur J Pharmacol. 2007;573:249252. 9 Nishi K, Inoue H, Schnier JB, Rice RH. Cyclin D1 downregula- tion is important for permanent cell cycle exit and initiation of differentiation induced by anchorage-deprivation in human kera- tinocytes. J Cell Biochem. 2009;106:6372. 10 Jerome-Morais A, Rahn HR, Tibudan SS, Denning MF. Role for protein kinase C- in keratinocyte growth arrest. J Invest Derma- tol. 2009;129:23652375. 11 Lehenkyi V, Beck B, Polakowska R, Charveron M, Bordat P, Skryma R, et al. TRPV6 is a Ca 2+ entry channel essential for Ca 2+ -induced differentiation of human keratinocytes. J Biol Chem. 2007;282:2258222591. 12 Koivisto L, Jiang G, Hakkinen L, Chan B, Larjava H. HaCaT keratinocyte migration is dependent on epidermal growth factor receptor signaling and glycogen synthase kinase-3. Exp Cell Res. 2006;312:27912805. 13 Prockop DJ, Kivirikko KI. Heritable diseases of collagen. N Engl J Med. 1984;311:376386. 14 Murray JC, Stingl G, Kleinman HK, Martin GR, Katz SI. Epider- mal cells adhere preferentially to type IV (basement membrane) collagen. J Cell Biol. 1979;80:197202. 15 Yang J, Meyer M, Muller AK, Bohm F, Grose R, Dauwalder T, et al. Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis. J Cell Biol. 2010;188:935952.