Construction of Targeting Vector and Generation of Sgk3 KO Mice
All animal experiments were conducted following institutional Committee on Animal Research Committee approval. The targeting strategy for disruption of the Sgk3 gene involved removing parts of exons 10, which contains the ATP-binding site necessary for the catalytic activity of SGK3, and 11, deleting intron 10, and introducing an in-frame STOP codon into exon 11. Plasmid pNTK loxp (gift from Dr. S. Coughlin, University of California, San Francisco, CA) was used to generate the targeting vector. Two mouse genomic fragments, containing exons 8–11 and exons 10–17, were amplified from 129 × 1/SvJ DNA by polymerase chain reaction (PCR) and cloned into pCR4-TOPO and pCR-XL-TOPO (both from Invitrogen, Carlsbad, CA), respectively, and characterized by restriction enzymes. The exon 8–11–containing construct was used as a template in a second round of PCR to generate a 2.6-kb exon 8–10 fragment with a BamHI site added to the 5′ end and an MfeI site added to the 3′ end. This fragment was used as the short arm and was inserted into the BamHI/MfeI sites of the targeting vector. The 10-kb-long arm fragment was generated by using the exon 10–17–containing construct as a template in a second round of PCR. A ClaI site was added to the 5′ end, and a STOP codon was added before the start of exon 11; a XhoI site was added to the 3′ end. This fragment was inserted into the ClaI/XhoI sites of the targeting vector. The targeting vector was linearized by digestion with XhoI and electroporated into RW-4 embryonic stem cells (derived from 129 × 1/SvJ mice). G418- and gancyclovir-resistant clones were initially screened by PCR using oligonucleotide primers located inside and outside the targeted locus to confirm homologous recombination. Two positive clones were expanded and their genomic DNA analyzed by Southern blot analysis after digestion by MfeI. An external probe (a 2-kb restriction fragment lying between exons 1 and 7) was used to verify correct targeting. The two positive clones were injected into C57BL/6 blastocysts and transferred into pseudopregnant females. Chimeric males, identified by their agouti coat color, were mated with C57BL/6 females. To generate mice homozygous for the targeted allele, the resulting Sgk3+/- males and females were interbred.
PCR Analysis of Genotype and Sex
Genomic DNA was prepared from tail biopsies by overnight digestion in 500 μl of proteinase K/STE (1% SDS, 50 mM Tris-Cl, pH 8.0, 0.5M NaCl, 1 mM EDTA) (0.5 mg/ml). Digests were diluted 1:100 and used directly in PCR reactions with the forward primer 5′CTTCTTGCAAAACGGAAACTGGATG3′ and the reverse primer 5′CCCCTCCATTAACAAAATCCAGAAC 3′. Reactions were performed using LA Taq
(TaKaRa; Otsu, Shiga, Japan) with the conditions 94°C 1 min; 94°C 30 s, 62°C 45 s, 68°C 7 min for 14 cycles, and then increased extension by 15 s/cycle; final extension of 72°C for 15 min. PCR products were resolved on 1% agarose gels. The wild-type (WT) allele PCR product was 0.2 kb; that for the mutated allele was 1.9 kb. Sexing of newborn mice was performed by PCR as described previously (McClive and Sinclair, 2001
Northern Blot Analyses
Total RNA was isolated using STAT-60 reagent (Tel-Test Inc., Friendswood, TX). Eight micrograms of RNA was resolved by formaldehyde-agarose gel electrophoresis, transferred to Hybond-NX membrane (Amersham Biosciences, Piscataway, NJ) and probed with a fragment spanning the entire Sgk3 open reading frame. After autoradiographic exposure, the membrane was stripped and reprobed multiple times by using fragments corresponding to the Sgk1, Sgk2, Akt1, Akt2 (to assess compensatory induction), and cyclophilin (loading control) coding sequences.
Western Blot Analysis
Western blot analysis was performed as described previously (Chen et al., 1999
). Protein extracts from WT and Sgk3
KO mice were used for Western blot analysis by using an SGK antibody (gift from Dr. G. Firestone, University of California, Berkeley, CA) that cross-reacts with SGK1, SGK2, and SGK3. To distinguish between the three SGK isoforms, SGK1, SGK2, and SGK3 proteins synthesized in a coupled reticulocyte system (Promega, Madison, WI) were analyzed on the same blot; this also confirmed the expected size for the SGK3 protein (56.4 kDa).
To analyze skin morphology, dorsal skin was biopsied and fixed overnight in 10% neutral buffered formalin (Fisher Scientific, Hampton, NH). Samples were dehydrated, paraffin-embedded, and sectioned (6 μm). For basic morphology, sections were deparaffinized and stained with hematoxylin and eosin; samples were taken from heterozygote and Sgk3 KO littermates to obtain sufficient numbers.
In Situ Hybridization
Localization of Sgk3 mRNA expression in dorsal skin was determined using 6-μm-longitudinal sections (paraffin-embedded) of skin from Sgk3+/–
mice by in situ hybridization, as described previously (Etchevers et al., 2001
), except sections were not treated with proteinase K, and after hybridization, slides were washed twice in 50% formamide, 1 × SSC, 0.1% Tween 20. Postpartum day 1 (P1), P3, and P4 sections were exposed to substrate for 9 d; P5 sections were exposed for 6 d. Sections were counterstained with nuclear fast red (Sigma-Aldrich, St. Louis, MO), dehydrated and permanently mounted.
Proliferating Cell Nuclear Antigen (PCNA) and β-Catenin Immunohistochemistry, and Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL)
Immunohistochemistry was performed on 6-μm-longitudinal sections (paraffin-embedded) from Sgk3 heterozygotye and KO mice by using the M.O.M. immunohistochemistry kit, and alkaline phosphatase detection system in conjunction with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (all from Vector Laboratories, Burlingame, CA) according to the manufacturer's protocol, except with incubation times of 60 min for primary antibody, 20 min for secondary antibody, and 20 min for ABC. Dilutions of 1:100 of anti-mouse PCNA antibody (Novocastra Laboratories, Newcastle-On-Tyne, United Kingdom), 1:50 of anti-mouse β-catenin antibody (BD Transduction Laboratories, Lexington, KY) were used. BCIP/NBT detection substrate was incubated for 20 to 80 min, to achieve optimal staining. Sections were counterstained with Nuclear Fast Red, dehydrated, and permanently mounted. TUNEL was performed using the DeadEnd colorimetric TUNEL system (Promega, Madison, WI) according to the manufacturer's protocol; sections were dehydrated and permanently mounted.
Cell Culture and [3H]Thymidine Incorporation in Mouse Primary Keratinocytes
Primary mouse keratinocytes were prepared from P4 heterozygote and KO mice by using a protocol developed in our laboratory. Briefly, culture flasks or plates first were coated with a collagen/fibronectin mix containing 0.01 mg/ml fibronectin, 0.032 mg/ml type 1 collagen and 0.1 mg/ml bovine serum albumin dissolved in mouse keratinocyte media (EMEM with EBSS; Cambrex Bio Science Walkersville, Walkersville, MD). Dispersed keratinocytes were collected by centrifugation and the cells then were resuspended in mouse keratinocyte media (EMEM with EBSS; Cambrex Bio Science Walkersville) containing 10% chelexed fetal bovine serum (FBS), penicillin/streptomycin/amphotericin, and 0.025 mM Ca2+. EGF (50 ng/ml) was added 5 h after initial plating, to allow optimum keratinocyte attachment. Primary keratinocytes (2 × 105) from Sgk3 heterozygote or KO mice were seeded on six-well plates and incubated for 24 h, followed by pulsing for 8 h with [3H]thymidine (1 mCi/well). Cultures were washed twice with phosphate-buffered saline, and reactions were terminated using ice-cold 5% trichloroacetic acid (TCA).Cells were washed with an additional volume of 5% TCA and with distilled water and were solubilized in 0.3 M NaOH for 10 min at 50°C. The amount of [3H]thymidine incorporated into DNA was determined by liquid scintillation counting.
Transient Transfection Assays
HaCaT cells (generously provided by Dr. N. Fusenig, German Cancer Research Center, Heidelberg, Germany) were maintained in DMEM supplemented with 10% FBS. Newborn human primary keratinocytes were maintained in medium 154CF supplemented with human keratinocyte growth supplement and 70 μM CaCl2 (Cascade Biologics, Portland, OR). The mouse SGK3 open reading frame was subcloned into pCDNA3 (SGK3), and expression vectors for kinase-dead (K191M, which prevents ATP-binding to the SGK3 catalytic site) and constitutively active (S486D, which substitutes an acidic residue that mimics phosphorylation by PDK1) forms of SGK3 were generated by PCR-based mutagenesis. The FKHRL1-responsive promoter plasmid (FHRE) and FKHRL1 expression plasmids were provided by Dr. M. Greenberg (Harvard Medical School, Boston, MA). The reporter consisting of seven Lef-1 response elements driving luciferase (p7 Lef-fos Luc) and a Lef-1 expression vector were provided by Dr. R. Grosschedl (University of Munich, Munich, Germany). For transient transfections, low passage HaCaT cells or primary keratinocytes at passage 3 or 4 were seeded 24 h before transfection at a density of 2 × 105 cells/well in six-well plates. Transient transfections were performed using the TransIT reagent (Mirus, Madison, WI) according to the manufacturer's protocol. For Lef-1 experiments in HaCaT cells, 100 ng of reporter, 10 ng of Lef-1 and 5, and 10 or 25 ng of mutant SGK3 were used. Twenty-four hours after transfection, serum-free medium was added; after a further 18 h, fresh serum-free medium with 50 μM LY294002 or dimethyl sulfoxide (vehicle) was added. Cells were harvested after a further 6 h and freeze-thaw lysed in 100 μl of 0.25 M Tris, pH 7.6. For human foreskin keratinocyte (NHK) cells, 100 ng of reporter, 100 ng of Lef-1, and 100 ng of mutant SGK3 were used. For FHRE experiments, 100 ng of FHRE, 300 ng of FKHRL1, and 25 ng of WT or mutant SGKs were used. For EGF treatment, 24 h posttransfection, insulin-free medium lacking EGF, or with 50 ng/ml EGF or transforming growth factor (TGF)-α, were added and cells harvested 24 h later. Luciferase activity of 5 μl of lysate was assayed using Promega Luciferin Reagent and normalized to total protein levels, which were measured by adding 100 μl of Bio-Rad protein assay dye (Bio-Rad, Hercules, CA) to 5 μl of lysate and measuring in a plate reader. Transfections were performed at least three times.
Weight and Growth Analyses
To assess for disturbances of weight and growth, several analyses were performed. Newborn mice from 7 litters were weighed within 18 h of birth, sacrificed and tails harvested, and genotype and sex determined by PCR as described. 10 d old mice from 7 litters were categorized according to whether they displayed the hair phenotype, and weighed; WT and heterozygote mice were not distinguished, and sex was not determined. Despite not distinguishing between sexes, this analysis is valid, because male and female mice of the same genetic background, including the B6;129 background, do not differ significantly in body weight at P10; body weights of male and female mice only begin to diverge after weaning (P21) (Rhees and Atchley, 2000
; Lupu et al., 2001
). Thus, differences in the numbers of male or female mice between genotypes will not skew the data. To calculate the relative weight of Sgk3
KO mice, their weights were expressed relative to the mean weights of WT and heterozygote mice (set to 100%). The relative weights of Sgk3
KO mice were calculated for seven litters and meaned. For growth curves from 3 to 8 wk of age, mice from 15 litters (including those weighed at P10) were sexed, genotyped by PCR, and weaned at 3 wk of age. Weights were measured weekly from 3 to 8 wk of age.
Intraperitoneal Glucose Tolerance Test
WT and Sgk3 KO littermates of the same sex were fasted overnight (16 h) and then injected intraperitoneally with 1 mg/g of body weight d-glucose [10% (wt/vol) stock solution in phosphate-buffered saline]. Blood samples were collected from the transversely sectioned tip of the tail, and whole blood glucose was measured using a Glucometer Elite (Bayer, Leverkusen, Germany) at 0 min (just before glucose injection), and at 15-, 30-, 60-, 90-, 120-, 180-, and 240-min intervals after the glucose load.
Sodium Balance Studies
Six each of wild-type and SGK3–/– mice (3 mo of age), were kept in metabolic cages for an acclimatization period of 4 d with free access to water and food (standard diet C1314, 0.2% Na; Altromin, Lage, Germany). Urine was collected every 24 h for 7 d and stored at –20°C. From the second day of urine collection, the standard diet was changed to a low sodium diet (C1036, 0.015% Na; Altromin). After 4 d, the mice were returned to the standard diet. Blood was drawn, from the orbital plexus before and after 4 d on the low sodium diet, and serum was separated by centrifugation and stored at –20°C. Serum aldosterone was measured with a commercial RIA-kit (Aldosterone RIA, Diagnostic Systems Laboratories, Webster, TX) according to the manufacturer's protocol. Urinary sodium concentration was determined using colorimetric analysis (Advia 1650; Bayer). Urinary 24-h excretion of sodium was calculated using the daily urine volume.
All data were analyzed using the Statview 4.5 software package. For growth curves, glucose tolerance tests and sodium balance studies, a repeated measure analysis of variance (ANOVA) was performed.