Alterations in mRNA expression and protein products following spinal cord injury in humans.
We examined the effects of spinal cord injury (SCI) on alterations in gene expression and respective protein products in human skeletal muscle 2 days and 5 days post-SCI. Biopsies were taken from skeletal muscle of 9 men and 1 woman (n = 10) (43.9 +/- 6.7 years) 2 days and 5 days post-SCI and from 5 healthy young men who served as controls (20.4 +/- 0.5 years). Global changes in gene expression were analysed using Affymetrix GeneChips on a subsample of subjects (n = 3). Candidate genes were then pursued via qRT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes. Immunohistochemistry (IHC) was used to localize proteins. Groups of transcripts showing the greatest percentage of altered expression, the most robust fold-changes, and indicative of involvement of an entire pathway using the GeneChip included genes involved in the ubiquitin proteasome pathway (UPP), metallothionein function, and protease inhibition. qRT-PCR analysis confirmed increases in gene expression for UPP components (UBE3C, Atrogin-1, MURF1, and PSMD11), the metallothioneins (MT1A, MT1F, MT1H), and the protease inhibitor, SLPI (P < 0.05) at 2 days and 5 days post-SCI. Protein levels of the proteasome subunit (PSMD11) and the metallothioneins were increased 5 days post-SCI. Protein levels of UBE3C, Atrogin-1, MURF1 and SLPI were unchanged (P > 0.05). IHC showed increased staining for PSMD11 and the metallothioneins 5 days post-SCI, along the peripheral region of the cells. IHC also showed altered staining for Atrogin-1 at 5 days post-SCI along the membrane region. Thus, there was a profound increase in gene expression of UPP components, the metallothioneins, and the protease inhibitor, SLPI, within 5 days of SCI. Increased protein levels for PSMD11 and the metallothioneins 5 days post-SCI, specifically along the cell periphery, indicate that proteins in this region may be early targets for degradation post-SCI.
Urso
Maria L
ML
Department of Exercise Science, University of Massachusetts, Amherst, MA 01003, USA. maria.urso@us.army.mil
Chen
Yi-Wen
YW
Scrimgeour
Angus G
AG
Lee
Patrick C
PC
Lee
K Francis
KF
Clarkson
Priscilla M
PM
D000328:Adult; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000473:pathology; D018482:Muscle, Skeletal / Q000473:pathology / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D053481:Secretory Leukocyte Peptidase Inhibitor / Q000235:genetics / Q000378:metabolism; D013119:Spinal Cord Injuries / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D025801:Ubiquitin; D008668:Metallothionein; D046988:Proteasome Endopeptidase Complex