Article |
Address correspondence to Sergei A. Grando, Dept. of Dermatology, University of California, Davis, 4860 Y St., #3400, Sacramento, CA 95817. Tel.: (916) 734-6057. Fax: (916) 734-6793. email: sagrando{at}ucdavis.edu
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Abstract |
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Key Words: integrins; cyclic GMP; cyclic AMP; knockout mice; acetylcholine
Abbreviations used in this paper: AC, adenylyl cyclase; ACh, acetylcholine; AGKOS, agarose gel keratinocyte outgrowth system; [Ca2+]i, intracellular-free calcium; cGMP, cyclic GMP; GC, guanylyl cyclase; GPCR, G proteincoupled receptor; IF, immunofluorescence; KC, keratinocyte; KGM, KC growth medium; KO, knockout; mAChR, muscarinic ACh receptor; PKA, protein kinase A; PKG, protein kinase G; ROK, Rho-associated protein kinase; siRNA, small interfering RNA; WT, wild-type.
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Introduction |
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To characterize the physiologic control of crawling locomotion of KCs, we developed an in vitro model of skin reepithelialization, termed agarose gel KC outgrowth system (AGKOS), which allows accurate evaluation of effects of pharmacologic compounds on lateral migration of KCs (Grando et al., 1993b). AGKOS measures a large cell population response (50,000 cells) in a milieu that approximates a physiologic one because KCs move over the ECM proteins produced and deposited on the dish surface by KCs during long-term incubations (Nickoloff et al., 1988; Marchisio et al., 1991). A shorter, 24-h assay that allows study of the initiation of KC outgrowth is a "scratch assay" (Savani et al., 1995). Compared with the AGKOS assay, it measures migration of a smaller, yet representative number of cells, i.e., up to 300 per a microscopic field at the magnification of 10.
Using AGKOS, we demonstrated that acetylcholine (ACh) is required to initiate outgrowth of KCs in vitro and that cholinergic drugs have profound effects on the motility of these cells (Grando et al., 1995; Zia et al., 2000). Human KCs functionally synthesize, store, release, and degrade ACh both in vivo and in vitro (Grando et al., 1993c). In the intact epidermis, KCs constantly move along an upward concentration gradient of ACh (Nguyen et al., 2001). The effects of ACh on cell motility are complex and include both stimulatory and inhibitory pathways regulating crawling locomotion (Zheng et al., 1994; Clancy and Abramson, 2000). Recent results strongly suggest that many of these effects are mediated by members of the muscarinic ACh receptor (mAChR) family (Varker and Williams, 2002; Chernyavsky et al., 2003). The mAChRs are prototypical members of the superfamily of G proteincoupled receptors (GPCRs; Caulfield, 1993; Wess, 1996). The M1, M3, and M5 mAChRs are preferentially coupled to activation of pertussis toxininsensitive G proteins of the Gq/11 family and stimulate phospholipase C, with resulting activation of PKC and the release of Ca2+ from intracellular stores. The other two mAChR subtypes, M2 and M4, are selectively coupled to pertussis toxinsensitive G proteins of the Gi/o class and mediate the inhibition of adenylyl cyclase (AC) activity and the activation of inward rectifier K+ channels. Stimulation of the M3 mAChR has been shown to up-regulate the binding activity of ß1 integrin, which is associated with increased cell adhesion to ECM proteins (Quigley et al., 1998; Williams, 2003) and inhibited cell migration (Varker and Williams, 2002). Although Gi signaling also alters integrin activity/expression, it leads to an up-regulated cell migration (Wang et al., 1999). Signaling from both G
q/11- and G
i/o-coupled mAChRs alters activity of the effector proteins of Rho family, with subsequent alterations in the Rho-associated protein kinase (ROK) activity (Harhammer et al., 1996). It has been demonstrated that the Rho family GTPases are essential downstream elements in the signaling pathway, linking mAChR stimulation to regulation of cell motility (Linseman et al., 2000).
The purpose of this study was to explore the role of KC M3 and M4 mAChRs in regulating KC migratory function and elucidate the responsible mechanism(s). We used a combination of three overlapping approaches to inhibit mAChR-coupled signaling pathways: (1) pharmacologic blockade with receptor antagonist and regulatory enzyme inhibitors; (2) mAChR gene silencing with small interfering RNA (siRNA); and (3) mAChR gene knockout (KO) in mice. We found that ACh stimulates the crawling locomotion of KCs by activating M4 receptors, but inhibits this activity through stimulation of M3 receptors. The M3 effects were mediated through the intracellular Ca2+-dependent guanylyl cyclase (GC)cyclic GMP (cGMP)protein kinase G (PKG) signaling upstream from the RhoROK pathway. The M4 effects resulted from inhibition of the inhibitory pathway ACcAMPprotein kinase A (PKA). The opposing mechanistic effects of M3 and M4 were accompanied by their reciprocal actions on the expression levels of specific integrins. These findings may aid the development of novel muscarinic drugs that are useful in facilitating wound healing.
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Results |
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When given alone, 10 nM MT3 reduced the migration distance of cultured human KCs by >50% (P < 0.05), whereas 4-DAMP did not produce any significant effect (P > 0.05), as could be expected when the two receptors, M3 and M4, which exhibit reciprocal effects on KC migration, are blocked simultaneously (Fig. 1 D).
To elucidate the role of mAChRs in regulation of KC migration speed, we used a scratch assay that measures predominantly linear migration (Savani et al., 1995). The results obtained through both assays were always coherent.
Effects of M3 and M4 mAChR gene KO on KC migration in vivo and in vitro
1-cm2 excisional wounds were inflicted in the back skin of M3/, M4/, and wild-type (WT) mice. The mice were killed 3, 5, 7, or 10 d later, and the rate of epithelialization was assessed in specimens of wound tissue stained with hematoxylin and eosin. The representative images used for quantitative histomorphometry, illustrating the time course of wound reepithelialization, are shown in Fig. 2 A. The morphometric analysis revealed that on the third day after wounding, M3/ mice exhibited a small increase (P > 0.05), whereas M4/ mice showed a significant (P < 0.05) decrease of epithelialization rate (Fig. 2 B). These changes became more pronounced on the fifth day after wounding, when the epithelialization rate in M3/ mice increased by 60% (P < 0.05) and that in M4/ mice decreased by
50% (P < 0.05).
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Taken together, these data indicated that the early stage of wound epithelialization, mediated by crawling locomotion of KCs (Woodley et al., 1993), was facilitated in the absence of M3 and delayed in the absence of M4 receptors.
To elucidate differences in hair follicle morphogenesis, a physiological process where massive KC migration occurs, we performed quantitative morphometric analysis of hair follicle development in mAChR KO mice. 1-d-old M3/ pups displayed accelerated hair follicle morphogenesis, as could be judged from a significantly (P < 0.05) increased number of hair follicles in advanced stages of perinatal follicle morphogenesis (stages 68) and a reciprocal decrease of the percentage of follicles at the early stages of their development (stages 13) compared with WT controls (Fig. 2, C and D). In marked contrast, the M4/ neonates showed a retardation of hair follicle morphogenesis evidenced by a significant (P < 0.05) increase in the number of hair follicles at the immature developmental stages and a corresponding reduction of the percentage of hair follicles at more mature stages (Fig. 2, C and D).
The roles of M3 and M4 mAChRs in regulating KC crawling locomotion were further investigated in AGKOS plates loaded with KCs grown from the epidermis of WT and M3/ and M4/ mutant mice. As expected, the lack of M3 receptors was associated with an increase of KC migration distance by 85% (Fig. 2 E). The M4/ KCs showed a significantly reduced migration distance (by >50%; P < 0.05). 10 nM of the M4-preferring antagonist MT3 significantly (P < 0.05) decreased the migration distances of M3/ KCs, but had no effects on the migration distance of M4/ cells (not depicted). 10 nM 4-DAMP increased the migration distance of M4/ KCs compared with nontreated M4/ cells (P < 0.05).
Role of intracellular-free calcium ([Ca2+]i) in the signaling pathways mediating reciprocal effects of M3 and M4 on KC migration
Because elevation of [Ca2+]i can mediate both M3 (Carroll and Peralta, 1998) and M4 (Ransom et al., 1991) signaling, we sought to determine if the inhibitors of Ca2+ ATPase in endoplasmic reticulum with thapsigargin or cyclopiazonic acid alter the stimulatory effect of siRNA-M3 and the inhibitory effect of siRNA-M4 on KC migration. The siRNA-M3 or siRNA-M4transfected versus intact KCs were exposed to either 1 µM thapsigargin (Fig. 3) or 20 µM cyclopiazonic acid (not depicted) dissolved in KGM for 30 min daily during the 10-d duration of the migration assay in AGKOS plates. Mobilization of [Ca2+]i produced a mild inhibitory effect on migration distance of intact KCs (P > 0.05), abolished an up-regulated migration of siRNA-M3transfected KCs (P < 0.05), decreasing it slightly below the normal level (P > 0.05), and produced no significant alterations of the down-regulated migration of siRNA-M4transfected cells (P > 0.05).
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Role of the ACcAMPPKA pathway in the mechanism of M4-dependent stimulation of KC migration
The immediate effect of M4 activation is inhibition of AC constitutively activated through Gs-coupled receptors, such as KC ß2 adrenergic receptors stimulated by autocrine epinephrine (Schallreuter, 1997). Activation of the cAMP signaling cascade is associated with inhibition of migration of various cell types (Johnson et al., 1972; McCawley et al., 2000). Hence, the functional inactivation of M4 that decreased KC migration could result from the inhibitory action of a Gs-coupled signaling pathway released from the M4 control by MT3 and siRNA-M4. The AC inhibitors up-regulated migration of siRNA-M4transfected KCs (P < 0.05), indicating that AC, rather than [Ca2+]i, plays a key role in the M4-coupled signaling pathway.
To elucidate the mechanism of M4 signaling in KCs downstream from inhibition of AC, we tested the effect of the cAMP analogue Sp-8-Br-cAMPS, which decreased the migration distance (P < 0.05; Fig. 3), suggesting further that M4 stimulates KC migration by putting a brake on the signaling pathway that includes the cAMP activation step. Because Sp-8-Br-cAMPS, just like naturally occurring cAMP, exhibits its biological action via activation of PKA (Seebeck et al., 2002), we sought to determine the role for PKA in the signaling pathway linking M4 to KC migration. 25 nM of the PKA inhibitor KT5750 increased the migration distance of siRNA-M4transfected KCs to the normal level (P > 0.05; Fig. 3). C3 exoenzyme and Y-27632 inhibited the migration of both of the KT5750-pretreated and siRNA-M4transfected KCs, indicating that in the signaling cascade linking M4 to stimulation of KC migration, the RhoROK pathway is situated distally from the cAMP-dependent events.
Relationship between the M3- and M4-coupled pathways of the physiologic regulation of KC migration by ACh
We tested the effect of 10 nM of the M4 blocker MT3 on the migration distances of KCs pretreated with Rp-8-pCPT-cGMP or KT5750. As seen in Fig. 3, although MT3 significantly (P < 0.05) decreased the migration distance of Rp-8-pCPT-cGMPpretreated cells, it had no effect on the KT5750-pretread KCs. Thus, when the M3-dependent inhibition of migration was blocked pharmacologically or due to the M3 gene silencing, crawling locomotion of KCs was up-regulated owing to their unopposed stimulation through the M4-coupled stimulatory pathway.
Effects of mAChR siRNAs on the integrin expression levels in human cultured KCs
Transfection of human foreskin KCs with siRNA-M3 resulted in a decrease of the relative amounts of 2 and
3 integrins by
50 and 40%, respectively (P < 0.05), and an increase in
5,
v, and ß5 expression by
30, 100, and 70%, respectively (P < 0.05; Fig. 4). KCs transfected with siRNA-M4 showed changes in the integrin expression pattern that were, in most cases, reciprocal to those observed in KCs transfected with siRNA-M3, suggesting that knockdown of M3 receptors favors the expression of the migratory integrins, whereas the silencing of M4 receptors leads to overexpression of sedentary integrins.
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To elucidate the role of PKA in the KC integrin shift, the cells were exposed for 6 h or 10 d to the PKA inhibitor KT5750, M4 antagonist MT3, or a combination of both. The short-term exposure produced no significant changes in the integrin phenotype (P > 0.05; Fig. 4). After the long-term incubation, which provided for KC migration, the cells exposed to KT5750 demonstrated slightly increased levels of the migratory integrins 5,
v, and ß5, which could be a result of the outside-in signaling characteristic of crawling KCs. The effect of a long-term exposure to MT3 was very similar to that of transfection with siRNA-M4 (Fig. 4) and included both up-regulation of the sedentary integrins
2 and
3, and down-regulation of the migratory integrins
5,
v, and ß5 (P < 0.05), which is consistent with migration inhibition by both MT3 and siRNA-M4 (Fig. 1 D). The cells exposed to both KT5750 and MT3 for 10 d had a decreased level of
3 and increased levels of
5 and ß5 (P < 0.05; Fig. 4).
Role of integrins in altered KC migration in mAChR KO mice
In WT mice, wounding down-regulated the expression of the sedentary integrins 2 and
3 in WT mice, as detected by Western blotting (Fig. 5 A) and semiquantitative IF (Fig. 5 B). In marked contrast, the protein levels of the migratory integrins
5,
v, and ß5 in wounded skin exceeded those found in the intact skin. In M3/ mice, the relative expression levels of all sedentary integrins were decreased in both intact and wounded skin, whereas those of migratory integrins were increased compared with WT mice (Fig. 5, A and B). In M4/ mice, the relative amounts of the sedentary integrins
2 and
3 were found to be significantly (P < 0.05) increased in the intact skin by both Western blotting and IF. Although wounding down-regulated expression of the sedentary integrins and up-regulated
5 and
V expression, the ß5 integrin levels were found to be below the control values in samples from both intact and wounded skin (Fig. 5, A and B). Although the relative amounts of the integrins under consideration in KCs residing in the epithelial tongues of WT versus mAChR KO mice were different, the immunostaining patterns were similar (Fig. 5 C).
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Finally, the role for integrins in providing a mechanistic link between M3 and M4 mAChR signaling pathways and crawling locomotion was investigated by measuring the rate of reepithelialization of in vitro wounds in the monolayers of WT and mutant KCs in the presence or absence of a specific anti-integrin antibody. Compared with WT KCs, the migration of M3 KO cells was significantly (P < 0.05) reduced in the presence of antibodies to migratory integrins, and that of M4 KO cells was reduced in the presence of antibodies to sedentary integrins (Fig. 5 E).
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Discussion |
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In this study, we observed a pronounced inhibition of KC crawling locomotion with the M4 receptorselective blocker MT3 (Jolkkonen et al., 1994). Moreover, the pivotal role of M4 receptors in facilitating KC crawling locomotion was convincingly demonstrated in experiments in which M4 receptor expression was either knocked down with siRNA or completely abolished in transgenic M4/ mice. Studies with anti-M3 siRNA and M3/ mice demonstrated that reduced or absent signaling through M3 receptors increases KC migration. Although the siRNA-M3 and siRNA-M4 used in this study specifically inhibited M3 and M4 expression, respectively, this technology does not allow permanent gene silencing. Hence, the changes in the migration distances observed in transfected cultures might represent the initial reduction in the M3 and M4 levels.
Because stimulation of migration of siRNA-M3transfected KCs could be suppressed with the M4 receptorpreferring antagonist MT3, the elimination of the inhibitory M3 receptor activity apparently unmasked the stimulatory activity of M4 receptors on the KC migratory function. It is well documented that other signaling molecules, such as growth factors, also play important roles in stimulating KC migration. Therefore, not surprisingly, compared with WT and M3/ mice, the epithelialization of M4/ mice was significantly delayed, but not completely blocked, as it would occur if M4 was the only signaling pathway regulating migration in KCs. The fact that wound epithelialization was significantly different 35 d after wounding reflects differences in KC migration because reepithelialization is predominantly mediated by the migratory function of KCs (Woodley, 1996).
In human epidermis, M4 receptors are predominantly expressed by the lowermost prickle cells that continuously migrate upward, whereas terminally differentiated KCs lack M4 receptors (Nguyen et al., 2001). In marked contrast, M3 receptors are predominantly expressed by basal KCs that establish hemidesmosomal contact with the basal membrane (Ndoye et al., 1998). Hence, M3 receptorinduced downstream signaling may support the establishment and/or maintenance of stable connections of KCs with the substrate in culture, and of the epidermis to the underlying dermis in the skin. It has been proposed that activation of M3 receptors is coupled to both up-regulation of ß1 integrinmediated cell adhesion (Quigley et al., 1998; Williams, 2003) and inhibition of cell migration (Varker and Williams, 2002). Therefore, the stimulation of KC migration caused by suppression of M3 receptor signaling might be mediated by decreased attachment of KCs to ECM proteins, such as laminin, which are known to decrease crawling locomotion (Decline and Rousselle, 2001).
The mechanism of muscarinic regulation of KC motility characterized in this study is schematically shown in Fig. 6. GPCRs play an important role in mediating biological effects of different hormones, growth factors, cytokines, and cytotransmitters on cell motility. Cell migration can be increased by activating a variety of heterotrimeric GPCRs, including those for adenosine (Woodhouse et al., 1998), angiotensin II (for review see Demoliou-Mason, 1998), lysophosphatidic acid (Lummen et al., 1997), opioids (Arai et al., 1997), sphingosine 1-phosphate (Graeler and Goetzl, 2002), thrombin (Arai et al., 1997; Lummen et al., 1997), and various peptide chemoattractants and cytokines (for review see Arai et al., 1997). Some of these GPCRs stimulate Gq/11, whereas others stimulate G
i/o, leading to specific biological responses (Lummen et al., 1997; for review see Demoliou-Mason, 1998). For instance, stimulation of mAChRs in oligodendrocytes activates
v integrin on the cell surface (Gudz et al., 2002).
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Results of this study strongly suggest that elevation of [Ca2+]i plays a role in the signaling cascade linking M3 to inhibited KC migration and that this cascade involves activation of the cGMP-dependent pathway. GC activation is mediated via an M3 mAChR coupled to a pertussis toxininsensitive G protein, whereas the GC inhibition can result from a pertussis toxinsensitive Gi/o proteincoupled mAChR (Tonnaer et al., 1991; Alfonzo et al., 1998). We have demonstrated previously that activation of M4 receptors results in a decrease in [Ca2+]i, whereas activation of M3 receptors increases Ca2+ in KCs (Nguyen et al., 2001), and that a nicotinic ACh receptormediated increase of [Ca2+]i is associated with decreased migration of human KCs in AGKOS (Zia et al., 2000).
To address a possibility that the two types of mAChRs, M3 and M4, exhibit reciprocal control of KC migration owing to competition of their effectors for common regulators of cell motility, Rho and ROK, we studied the effects of Rho and ROK inhibitors on the migration of intact and siRNA-M3 or siRNA-M4transfected KCs in AGKOS plates. The obtained results indicate that regulation of the RhoROK pathway is a common endpoint in the signaling mechanism, mediating muscarinic control of KC migration. Because cAMP inhibits activation of Rho (Gratacap et al., 2001), mostly owing to its PKA-dependent phosphorylation (Dong et al., 1998), one of the mechanisms mediating the stimulatory effect of M4 signaling on KC migration, in addition to inactivation of GC, could be inhibition of the cAMP cascade resulting in PKA activation. This supposition was corroborated by the ability of the PKA inhibitor KT5750 to reverse inhibited migration of siRNA-M4transfected or MT3-treated KCs. Thus, the stimulatory effect of M4 on KC migration stems from M4-dependent inhibition of AC activation through a Gs protein coupled by one of the KC GPCRs that are known to inhibit KC migration, such as ß2-adrenergic receptors that can inhibit KC migration via ß2-adrenergic receptormediated elevation of cAMP (Donaldson and Mahan, 1984).
In conclusion, the available data indicate that KC migration is regulated by ACh through both stimulatory and inhibitory signaling pathways, involving different populations of cell surface ACh receptors. The diversity of ACh receptors expressed by KCs in the course of their maturation would allow ACh to exert different effects at various stages of cell development. We propose that simultaneous stimulation of KC M3 and M4 receptors by endogenously secreted ACh is required to synchronize the development of ionic and metabolic events in migrating KCs. These findings offer novel mechanistic insights underlying the cholinergic control of KC crawling locomotion. They also suggest that mAChR-selective drugs and siRNA may serve as novel tools to modulate abnormal KC migration, such as in wounds that fail to heal.
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Materials and methods |
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mAChR mutant mice
Mice deficient in M3 or M4 mAChRs (M3/ and M4/ mice, respectively) were generated as described previously (Gomeza et al., 1999; Yamada et al., 2001). In all experiments, age- and gender-matched WT mice of the same genetic background (129/SvEv x CF1) were used as controls. Mouse genotyping was performed by PCR analysis of mouse tail DNA.
Cultures of human and murine KCs
Human KCs were isolated from foreskin epidermis and grown in culture as detailed elsewhere (Grando et al., 1993a). Murine KCs were similarly isolated from the epidermis of WT and M3/ and M4/ mutant mice that were 12 d old and grown using the cell culture techniques optimized for mouse KCs (Arredondo et al., 2002).
siRNA preparation and KC transfection
To design target-specific siRNA duplexes, we selected sequences of the type AA (N19) UU (N, any nucleotide) from the open reading frame of the target mRNAs to obtain a 21-nt sense and 21-nt antisense strand with symmetric 2-nt 3' overhangs of identical sequence (Elbashir et al., 2001). The siRNA-M3 and siRNA-M4 were custom synthesized by Dharmacon. The target sequences for the human CHRM3 (NM_000740) and CHRM4 (NM_000741) genes were as follows: CHRM3, 5'-GGTCAACAAGCAGCTGAAG-3' (corresponding to amino acids 280300); and CHRM4, 5'-CCTCTACACCGTGTACATC-3' (corresponding to amino acids 259279). The negative control siRNA-targeting luciferase gene was also purchased from Dharmacon. For transfection, human KCs were seeded at a density of 5 x 104 cells per well of a 24-well plate, and incubated for 1624 h to achieve 70% confluence. To each well, increasing concentrations (see Results) of siRNA duplex in the transfection solution were added, and the transfection was continued for 16 h at 37°C in a humid, 5% CO2 incubator. On the next day, the transfection medium was replaced by KGM, and the cells were incubated for 72 h to achieve maximum inhibition of mAChR protein expression. After 72 h of incubation, the cells were harvested and the proteins were extracted and used in Western blotting assays of integrin expression. The siRNA transfection efficiency in KCs was also assayed using FITC-labeled luciferase GL2 duplex (Dharmacon) with the target sequence 5'-CGT ACG CGG AAT ACT TCG A-3'.
In vitro KC migration assays
AGKOS assay.
Second passage human or murine KCs were suspended in KGM, counted in a hemocytometer, loaded at a high density (4 x 104 cells/10 µl) into each 3-mm well in an agarose gel, and used in AGKOS assays as detailed elsewhere (Grando et al., 1993b; Zia et al., 2000). KCs were fed with KGM containing various concentrations of cholinergic drugs or no drugs (control), and incubated for 10 d in a humid CO2 incubator with daily changes of medium. Some human KCs were first transfected with siRNA, and then exposed to drugs. To standardize results obtained in experiments using KCs from different donors, the mean values of the migration distances were converted into the percentage of control. The control value for KCs from each particular donor was determined by measuring the baseline migration distance (in millimeters) and taken as 100%. In parallel studies, to control for changes in the rate of KC proliferation on the migration distances measured by AGKOS assay, we exposed KCs in AGKOS plates to test compounds in the presence of 10 µg/ml of the growth-arresting agent mitomycin C (Sigma-Aldrich).
Scratch assay.
The original scratch assay (Savani et al., 1995) was adapted to measuring KC migration as detailed elsewhere (Cha et al., 1996). In brief, KCs were either grown to confluence or freshly seeded in 6-well tissue culture dishes. Some dishes were coated with fibronectin, laminin, collagen I or IV (all from BD Biosciences), or vitronectin (Sigma-Aldrich). The monolayers were scratched with a 100-µl pipette tip and incubated at 37°C and 5% CO2 in air until complete reepithelialization of wounded monolayers occurred in one of the cultures, but no longer than 24 h. To inhibit proliferation, for the first 2 h of incubation, KCs were fed with KGM containing 10 µg/ml mitomycin C. Reepithelialization was documented by photography and the amount of migration was quantitated by computer-assisted image analysis software (IP Lab; Scanalytics). The residual gap between KCs migrating toward each other from the opposing sides of the in vitro wound, i.e., the area of the scratch remaining unfilled, was measured and the results were expressed as percentage of reepithelialization determined in control, nontreated monolayer. Each experiment was performed in triplicate.
In vivo wounding and morphometric assay of epithelialization rate
Because the wound healing process is dependent on the stage of the hair cycle, in each experiment we used the mice of exactly the same age and gender with hair cycles that were synchronized using the anagen induction technique (Paus et al., 1990). In brief, 67-wk-old WT and M3/ and M4/ mutant mice in the state of telogen, as judged by their pink skin color, were anesthetized and stripped of hair using a wax and rosin mixture that was painted over the back of the mouse and after hardening was peeled off, inducing the resting follicles to enter anagen. 5 d later, full thickness excisions through the panniculus carnosus were made on the anesthetized skin using a uniform square template with a side length of 1 cm. Each animal received two wounds at the symmetric sites of the central back, 0.5 cm off the vertebral line. Wounds were left undressed and wounded animals were individually housed under aseptic conditions for 3, 5, 7, and 10 d, after which time the mice were killed by a CO2 overdose. Equally sized pieces of the wounded and intact skin near the wound were excised. To optimize the yield of migrating KCs, we always harvested the wound border by shaving a narrow strip along the perimeter of the wound. For extraction of total proteins used in Western blotting assay, the panniculus carnosus was removed (confirmed microscopically), and tissue was weighed, diced, and sonicated. At least six animals per time point and genotype were used. The morphometric analysis on sections was always performed from the middle of the wound. The rate of epithelialization was assayed in hematoxylin and eosinstained cryostat sections of the wound tissue by measuring the lengths of the tongues of new epithelium extending from either side of the wound, in accordance with standard protocols of morphometric analysis (Graves et al., 2001). In brief, three to five random microscopic fields were captured at a magnification of 4 in each skin section cut perpendicularly to the edge of the wound. The images were printed and the rate of epithelialization was computed by measuring directly on the prints the length of the epithelialization tongue advancing over the wound bed from its margin, which could be easily identified based on the abrupt change in the appearance of dermal ground substance at the excision line (Fig. 2 A). In each image, the length of epidermal outgrowth was measured in millimeters.
Morphometric analysis of hair follicle morphogenesis in mAChR KO mice
The morphometric analysis was performed in the longitudinal sections of dorsal skin of 1-d-old M3/ or M4/ mice versus WT littermates. The eight defined substages of hair follicle morphogenesis were identified based on the criteria defined by Paus et al. (1999). The percentage of hair follicles in each stage of morphogenesis was assessed using the established technique detailed elsewhere (Botchkarev et al., 1998). 100 follicles in each of the 50 microscopic fields (at a magnification of 10) of vertical skin sections derived from at least four different animals were calculated in each strain.
IF assay
The IF experiments were performed as detailed previously (Ndoye et al., 1998), using freshly frozen specimens of intact and wounded murine skin or cultured KC monolayers as a substrate, and computer-assisted image analysis with a software package purchased from Scanalytics. In tissue samples, the intensity of fluorescence was calculated pixel by pixel by dividing the summation of the fluorescence intensity of all pixels by the area occupied by the pixels (i.e., segment), and then subtracting the mean intensity of fluorescence of a tissue-free segment (i.e., background). For each specimen, a minimum of three different segments in at least three different microscopic fields were analyzed. The specimens were examined with a fluorescence microscope (model Axiovert 135; Carl Zeiss MicroImaging, Inc.).
Western blotting assay
Proteins were isolated by adding 1.5 ml isopropyl alcohol per 1 ml TRIzol reagent (GIBCO BRL) to the phenolethanol supernatant of homogenates of the samples of human and murine KCs, washed, dissolved in a sample buffer, separated via 415% SDS-PAGE, and electroblotted onto a 0.2-µm nitrocellulose membrane (Bio-Rad Laboratories). The membranes were developed using a chemiluminescent detection system (ECL Plus; Amersham Biosciences) and scanned (StormTM/FluorImager; Molecular Dynamics). The relative density of scanned bands was determined by area integration using ImageQuant software (Molecular Dynamics), and the results were expressed as integrated intensity of pixels of the spot excluding the background. The final results were expressed as ratios of the densitometry value of each integrin to that of ß-actin in the same lane, compared with the values obtained in control samples. The protein content ratio in each control sample was always set equal to 1.
Statistics
The results of quantitative experiments were expressed as means ± standard deviation. Significance was determined using Student's t test.
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Acknowledgments |
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Submitted: 9 January 2004
Accepted: 9 June 2004
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References |
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