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Increased expression of Ero1L-alpha in healing fetal wounds
© Kathju et al; licensee BioMed Central Ltd. 2011
Received: 15 November 2010
Accepted: 6 June 2011
Published: 6 June 2011
Adult mammalian tissues heal injury to the skin with formation of scar; this process quickly seals an injured area, however, excessive scar formation can become a source of persistent pathology, interfering with multiple vital functions. In contrast, mammalian fetal tissue can heal without scar formation. We previously sought to model scarless healing in a rabbit fetal skin wound and identified gene products differentially expressed during fetal wound healing through PCR suppression subtractive hybridization (PCR SSH). One of these transcripts, previously identified simply as clone 11, showed putative increased expression in wounded fetal skin. This study establishes its identity as Ero1L-alpha and confirms its elevated expression in healing fetal wounds.
After obtaining further sequence by 5' rapid amplification of cloned ends (RACE) we find that clone 11 is Ero1L-alpha. We determined that clone 11, a differentially expressed transcript in fetal wound healing, comprises the 3' untranslated region (UTR) of an approximately 4 kb transcript in rabbit tissues that corresponds to Ero1L-alpha. We showed that Ero1L-alpha is expressed predominantly as two transcripts in rabbit skin, namely a 1.6 kb transcript and the 4.0 kb transcript recovered in our PCR SSH screen via its 3' UTR sequence. However, a third transcript of 2.9 kb was also detected in Northern blots and was subsequently cloned and confirmed by 3' RACE. Knockdown of the clone 11 sequence in rabbit adult fibroblasts via siRNA resulted in significantly decreased Ero1L-alpha message expression. Increased expression of clone 11 (Ero1L-alpha) in a variety of cell types during the wound healing response was also confirmed by in situ hybridization.
Ero1L-alpha is one of the previously unknown clones identified in a PCR SSH screen for genes differentially expressed in fetal wounded tissue. In situ hybridization confirms that Ero1L-alpha shows increased expression in multiple cell types after wounding of the fetal integument.
Mammalian skin has multiple critical functions including providing homeostasis and serving as a first line of defence against infection. When injury to the skin occurs, a complex series of processes initiate its repair [1, 2]. Adult (postnatal) mammals heal injury to the skin with attendant scar formation [3–5]; this process quickly seals an injured area, but excessive scar formation can become a source of persistent pathology and can interfere with numerous vital functions. In contrast, mammalian fetal skin can heal without scar formation. Much research has focused on identifying the mechanisms underlying scarless fetal wound repair; these experiments have primarily compared adult and fetal wound healing by examining various growth factors/cytokines, extracellular matrix proteins, and chaperonins [6–13]. To date, no specific critical pathways determinative for scarless repair have been established.
We have previously examined scarless wound healing in fetal skin by incisional wound modelling in rabbits . Using PCR suppression subtraction hybridization (PCR SSH) we identified transcripts exhibiting differential expression during the fetal wound healing response. Because PCR SSH compares two conditions across the entire expressome and recovers only fragments of gene products, numerous genes of unknown identity and/or function were recovered. One of the unidentified gene products, designated clone 11, was upregulated in pooled samples of fetal wounded tissue samples 12 hours post-wounding when compared to fetal unwounded control skin tissue. Herein, we present evidence identifying this transcript as Ero1L-alpha and confirming its elevated expression with in vivo studies.
Materials and methods
Source Tissues and Fibroblast Culture
All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC); details of animal sample collection were as described in Kathju et al. ~1 cm incisional wounds were placed on the dorsums of fetal rabbits at 20-21 days gestation, then harvested 8 days later (as well as unwounded fetal control skin). For in situ hybridization studies, fetal wounded and unwounded control tissues were stored in 10% Neutral Buffered Formalin (up to 7 days) before embedding; processing, paraffin embedding, and sectioning of samples were performed by Research Histology Services (Pittsburgh, PA) using standard conditions.
Primers, probes, siRNAs used
(from Figure 2A, if applicable)
clone11 siRNA sense
clone11 siRNA antisense
scrambled siRNA sense
scrambled siRNA antisense
Ero1LA real time for
Ero1LA real time rev
Ero1LA real time probe
clone11 real time for
clone11 real time rev
clone11 real time probe
GAPDH real time for
GAPDH real time rev
GAPDH real time probe
RNA extraction/purification of samples
For all tissue culture RNA purifications, total RNA was obtained using the RNeasy Micro Kit (Qiagen Inc. USA, Valencia, CA) following manufacturer's protocols with a DNase treatment step. The quality of total RNA extracted from fetal and adult tissue and fibroblasts was examined by capillary electrophoresis using an Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Palo Alto, CA), and the quantity determined using the OD260/OD280 ratio measured using a ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE).
Rapid amplification of cloned ends (RACE) was used to obtain 3' UTR sequence for the various Ero1L-alpha clones. The GeneRacer kit with AMV reverse transcriptase (Invitrogen) was used for 3' RACE following manufacturer's directions; 2 μg of total RNA from fetal control skin was used as the source RNA. All subsequent PCR was done using the AccuPrime HF PCR system (Invitrogen) and using the primers listed in Table 1 following manufacturer's directions. Cloned amplimers from RACE reactions were then sequenced.
Primers for Ero1L-alpha were designed from the predicted rabbit sequence for Ero1L-alpha (ENSOCUG00000012632) from Ensembl  and primers for GAPDH were designed from the NCBI rabbit GAPDH sequence (NM_001082253) .
DIG RNA probes
DNA constructs for DIG RNA probes were prepared by either DNA digests of plasmids bearing subcloned inserts or by direct PCR of cloned and sequenced probes; DNA templates were gel extracted before use. DIG RNA probes were prepared using the DIG RNA labelling kit (Roche, Indianapolis, IN) and T3 RNA Polymerase (Promega Corporation, Madison, WI). RNA was purified using the RNeasy Micro Kit (Qiagen) following manufacturer's protocols.
Northern blots were either commercially prepared (Zyagen, San Diego, CA) or prepared using passage three fetal and adult fibroblast RNA. Northern blots were prepared using reagents from the NorthernMax kit (Applied Biosystems/Ambion, Austin, TX) and with the NorthernMax Loading Dye without ethidium bromide. Total RNA was used as the source for preparing mRNA using the Oligotex kit (Qiagen). 100 nanograms of mRNA and RNA marker (Invitrogen) were separated on 1% denaturing formaldehyde gels and transferred to a positively charged nylon membrane (Roche). RNA was cross-fixed to the membrane using a UV light box (SpectroLinker XL-1000, optimal cross-link settings).
Blots were rinsed with DEPC-treated water, and then prehybridized in DIG EasyHyb buffer (Roche) following manufacturer's protocol. Blots were hybridized overnight at 68°C in DIG EasyHyb with 100 ng DIG-labeled probe. Blots were washed for two 5 minute low stringency washes in 2 × SSC, 0.1% SDS at room temperature followed by two 20 minute high stringency washes in 0.2 × SSC, 0.1% SDS at 68°C. All blocking, washing and detection of DIG were done with the DIG wash and Block kit (Roche) and CDP-Star (Roche) following manufacturer's protocols.
Quantitative real time RT-PCR
The primer sets for rabbit clone 11 and rabbit Ero1L-alpha were designed using the initial sequence for clone 11 obtained by PCR SSH, plus additional sequence obtained through 5' RACE and 3' RACE, as well as the predicted rabbit sequence for Ero1L-alpha (ENSOCUG00000012632) from Ensembl . The Taqman primers/probes reported in Table 1 were designed using Primer Express software (Applied Biosystems, Foster City, CA). Initial RT-PCR assays on 100 ng of fetal unwounded control RNA were used to verify that each of the primer sets was detectable in fetal tissue and resulted in only a single amplicon of the expected molecular weight. Primer sequences for the rabbit GAPDH (used as an internal control) were previously published . All primers and fluorocoupled Taqman probes were purchased from Integrated DNA Technologies (Coralville, IA).
100 ng of total RNA from samples was used for reverse transcriptase (RT) reaction (using gene-specific reverse primer and 10 μl of total volume); for subsequent real time PCR assays, 1.5 μL of RT reaction, 800 nM of each primer, and 160 nM of the appropriate probe (final concentrations) in a total volume of 15 μl were used. The remaining protocol parameters for RT reaction and real time PCR were followed as previously described . Using the comparative critical cycle (Ct) method and using GAPDH as the endogenous control, the expression levels of the target genes were normalized and the relative abundance was calculated. Results shown are representative of three independent experiments performed in triplicate; statistical analysis for significance was performed using a Student's t-test.
in situ Hybridizations
Sectioned slides of unwounded fetal skin or wounded fetal skin at 8 days post-injury were processed as described , except using paraffin embedded sections. Slides were deparaffinated using three xylene washes and passed through an ethanol series, with a final wash in 1× PBS. Slides were then processed as described , using a 48 hour hybridization at 58°C. Slides were counterstained with 0.1% Nuclear FastRed (Vector Labs, Burlingame, CA) and mounted with aqueous ImmuMount (ThermoFisher, Pittsburgh, PA) before photographing.
We originally obtained only several hundred base pairs of clone 11 sequence from our PCR SSH screen. We then undertook both 5' and 3' RACE to obtain further contiguous clone 11 sequence in the hopes of establishing its identity. Using the Ensembl rabbit database and BLAST, we determined that this expanded clone 11 sequence was derived from a rabbit genomic region approximately 2.5 kb from the predicted gene Ero1L-alpha. In humans, two transcripts of Ero1L-alpha are known to exist; both contain a 1290 bp coding region but differ in the length of their 3' UTRs, with the longer one approximating 5.3 kb in total length.
We therefore hypothesized that clone 11 in the rabbit was the extended 3' UTR form of Ero1L-alpha. Using the rabbit predicted Ero1L-apha sequence from Ensembl (ENSOCUG00000012632), we designed primers to amplify (by RT-PCR) a large portion of the Ero1L-alpha coding region from rabbit fetal control RNA. This amplimer was subcloned and sequenced and matched the predicted rabbit sequence from Ensembl, with 92% sequence similarity to human Ero1L-alpha and 97% predicted protein similarity within the coding region of Ero1L-alpha.
Using a DIG-labelled probe derived from rabbit Ero1L-alpha coding sequence, we sought to determine which transcript isoforms are expressed in fetal and adult skin tissues and fibroblasts, and to confirm that Ero1L-alpha is expressed as two major transcripts in the rabbit as was previously reported in humans. Using a Northern blot prepared with total RNA isolated from fetal and adult unwounded skin tissues and from fetal and adult fibroblasts in culture, two transcripts of the expected molecular weights (~1400 bp and ~ 4 kb) were in fact observed in all samples (Figure 2B). Interestingly and surprisingly, a third minor message isoform corresponding to some 2.9 kb in length was also detected. We have subsequently used 3' RACE (using primers within the Ero1L-alpha coding sequence) to amplify and subclone the 3' end of this isoform, and confirm that it carries coding sequence identical to the other two transcripts, but with an intermediate length 3' UTR (data not shown). A full schematic, showing the various Ero1L-alpha message isoforms, together with locations of probes and primers is depicted in Figure 2A.
Ero1L-alpha plays a major role in the oxidative protein folding pathway in the endoplasmic reticulum, enabling proteins to form disulfide bonds , and is specifically up-regulated in response to hypoxic conditions . It has not been previously implicated in wound healing, but it is not surprising that its expression may be elevated in response to some hypoxic or ischemic stimulus at the zone of injury. It is interesting that we recovered only the 4 kb variant of Ero1L-alpha transcript in our PCR SSH screen, with its attendant lengthy 3' UTR. Such UTR sequences have been found in other systems to regulate gene expression through post-transcriptional mechanisms [21–24], and it may be that a similar phenomenon is occurring here. More study will be required to fully explicate the specific functional and regulatory significances of the multiple Ero1L-alpha isoforms we have identified.
We conclude that one of the previously unidentified gene fragments recovered in a PCR SSH screen examining fetal wound healing, clone 11, is a 4 kb isoform of Ero1L-alpha, featuring an extended 3' UTR sequence. Ero1L-alpha is expressed as three distinct mRNA isoforms in fetal and adult skin tissues and fibroblasts, including a novel 2.9 kb variant. The 4 kb isoform of Ero1L-alpha shows increased expression in multiple cell types after fetal integumentary wounding by in situ hybridization.
This study was supported by the Allegheny Singer Research Institute, Allegheny General Hospital and Pittsburgh Tissue Engineering Institute (PTEI). This work was funded from the grants awarded to S.K. (DE 014780), and L.S. (3M Fellowship). We thank Dr. Melissa Gallo for helpful discussions and critical reading of the manuscript. We extend our thanks to Ms. Mary O'Toole for her assistance in preparing this manuscript.
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