A Transcriptome Study of Progeroid Neurocutaneous Syndrome Reveals POSTN As a New Element in Proline Metabolic Disorder
Huang Yu-Wen1,2, Chiang Ming-Fu3,4,5, Ho Che-Sheng6, Hung Pi-Lien7, Hsu Mei-Hsin7, Lee Tsung-Han2, Chu Lichieh Julie8, Liu Hsuan8,9, Tang Petrus10, Victor Ng Wailap1,11,12,*, Lin Dar-Shong2,6,13,*
1Institute of Biotechnology in Medicine and Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming University, Taipei, Taiwan. 2Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan. 3Department of Neurosurgery, Mackay Memorial Hospital, Taipei, Taiwan. 4Mackay Junior College of Medicine, Nursing and Management, Taipei, Taiwan. 5Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei, Taiwan. 6Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan. 7Department of Pediatric Neurology, Kaohsiung Chang Gung Memorial Hospital, and Chang Gung University College of Medicine, Kaohsiung, Taiwan. 8Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan. 9Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 10Molecular Regulation and Bioinformatics Laboratory and Department of Parasitology, Chang Gung University, Taoyuan, Taiwan. 11Institute of Biomedical Informatics and Center for Systems and Synthetic Biology, National Yang Ming University, Taipei, Taiwan. 12Department of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan. 13Department of Medicine, Mackay Medical College, New Taipei, Taiwan
Aging is a complex biological process. A study of pyrroline-5-carboxylate reductase 1 (PYCR1) deficiency, which causes a progeroid syndrome, may not only shed light on its genetic contribution to autosomal recessive cutis laxa (ARCL) but also help elucidate the functional mechanisms associated with aging. In this study, we used RNA-Seq technology to examine gene expression changes in primary skin fibroblasts from healthy controls and patients with PYCR1 mutations. Approximately 22 and 32 candidate genes were found to be up- and downregulated, respectively, in fibroblasts from patients. Among the downregulated candidates in fibroblasts with PYCR1 mutations, a strong reduction in the expression of 17 genes (53.1%) which protein products are localized in the extracellular space was detected. These proteins included several important ECM components, periostin (POSTN), elastin (ELN), and decorin (DCN); genetic mutations in these proteins are associated with different phenotypes of aging, such as cutis laxa and joint and dermal manifestations. The differential expression of ten selected extracellular space genes was further validated using quantitative RT-PCR. Ingenuity Pathway Analysis revealed that some of the affected genes may be associated with cardiovascular system development and function, dermatological diseases and conditions, and cardiovascular disease. POSTN, one of the most downregulated gene candidates in affected individuals, is a matricellular protein with pivotal functions in heart valvulogenesis, skin wound healing, and brain development. Perturbation of PYCR1 expression revealed that it is positively correlated with the POSTN levels. Taken together, POSTN might be one of the key molecules that deserves further investigation for its role in this progeroid neurocutaneous syndrome.
Huang Yu-Wen,Chiang Ming-Fu,Ho Che-Sheng, et al. A Transcriptome Study of Progeroid Neurocutaneous Syndrome Reveals POSTN As a New Element in Proline Metabolic Disorder[J]. Aging and disease,
2018, 9(6): 1043-1057.
Figure 1. Intracellular proline levels of ARCL2B patient primary skin fibroblasts are lower than healthy controls. (A) Overview of proline biosynthesis. Proline is synthesized via glutamate and ornithine routes. The enzymes P5CS and OAT catalyze the conversion of glutamate and ornithine, respectively, to P5C which is subsequently converted to proline by PYCR1 and PYCR2. (B) Intracellular proline levels in the fibroblasts from healthy controls and patients with PYCR1 mutations (n=4). P5C: Δ1-pyrroline-5-carboxylate; GLS: Glutaminase; GAD: Glutamate decarboxylase; GABA: γ- aminobutyric acid; P5CS: P5C synthase; P5CDH: P5C dehydrogenase; OAT: Ornithine aminotransferase; POX: Proline oxidase; PEPD: Proline dipeptidase; ***: p < 0.001
4 & 6
4 & 5
Lax wrinkled skin
Typical facial gestalt
Aortic root dilatation Sinus of Valsalva (mm) (Range) Z-Score
+ + 23.6 (13.56-20.81) 3.47
- 19.8 (14.16-21.71) 0.97
- 19.1 (16.59-25.55) -0.86
+ 22.5 (14.05-21.54) 2.46
TR, MR, RAA
Abnormal brain MRI
Clinical features (neonatal)
Thin, translucent skin
Postnatal growth delay
Late fontanel closure
Table 1 Clinical features manifested in four ARCL2B patients with PYCR1 mutations.
Fold Change (Patients/ Controls)a
Genes downregulated in patients
Matrix metallopeptidase 1
Prostaglandin-endoperoxide synthase 2
EGF containing fibulin like extracellular matrix protein 1
Insulin like growth factor binding protein 3
Cytochrome p450 family 1 subfamily B member 1
Serine/threonine kinase 32B
ADAM metallopeptidase domain 12
SPARC/osteonectin, cwcv and kazal like domains proteoglycan 1
CCAAT/enhancer binding protein delta
Collagen type III alpha 1 chain
ADAM metallopeptidase with thrombospondin type 1 motif 5
Leucine rich repeat containing G protein-coupled receptor 4
Cornichon family ampa receptor auxiliary protein 3
Histone cluster 1 H4 family member h
Ankyrin repeat domain 30B like
Adhesion g protein-coupled receptor E5
G-protein coupled receptor
Table 2 List of 54 differentially expressed gene candidates in ARCL2B patients’ primary skin fibroblasts.
Figure 2. PYCR1 mutations affected its mRNA and protein levels in patients’ primary skin fibroblasts. (A) Immunoblot showing the truncated PYCR1 protein p.P115fsX7 is undetectable in fibroblasts from patients (P2 and P3) with homozygous c.345delC mutation, whereas the mutant proteins p.G248E (P1) and p.A187T (P4) are detected in samples with heterozygous mutations. (B) PYCR1 levels relative to GAPDH in four each of healthy control and patients were determined by SYBR Green based qRT-PCR. Data is expressed as mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (C) Different single amino acid substitutions in P1 and P4 impeded the PYCR1 half-life as determined by cycloheximide (CHX) chase assay. Cells were harvested and lysed at 0, 4, 8, 16, and 24 h after treatment in medium containing 20 µg/ml CHX. (D) Results in (C) were quantified using the IMAGE J software. α-tubulin was used to normalize the PYCR1 protein levels. The relative levels at 0 hr were defined as 1 for each sample.
Figure 3. qRT-PCR validated the expression changes of PYCR1 affected extracellular space protein genes. (A) Relative change of the most up- and downregulated expressed genes as determined by RNA-Seq analysis. (B) qRT-PCR validation of the two candidates in (A). (C) Gene ontology (GO) analysis shows enrichment of genes related to extracellular marix/region components are downregulated in patients’ fibroblasts. (D) The expression levels of ten extracellular space protein genes (fold-change > 2, TPM > 10) in the skin fibroblasts of all four patients and three different healthy donors were quantified by qRT-PCR. Data is expressed as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01.
Figure 4. IPA analysis indicated differentially expressed gene candidates are associated with cardiovascular, ophthalmic, and dermatological diseases. Genes with fold change > 2 and TPM > 10 were analyzed. (A) Genes implicated in the principal biological function categories related to the catalogues “Diseases and Disorders” and “Physiological System Development and Function”. (B) List of the genes implicated in top five catalogues. The input putatively up- and downregulated genes are indicated by red and green texts, respectively. The molecules located in extracellular space are bold and underlined.
Figure 5. Perturbations of PYCR1 expression modulate the expression of extracellular space genes in skin fibroblast. (A) Western blot (left) and quantification (right) of PYCR1 and POSTN protein levels in primary skin fibroblasts from controls and patients. Data was expressed as means ± SD from four biological replicates. (B) Evaluation of knockdown efficiencies of pLKO.1 lentiviruses expressing five independent PYCR1 shRNAs in primary skin fibroblasts. At 48 h post-transduction, skin fibroblasts were collected for RNA isolation and qRT-PCR. (C) At 72 h post-transduction, proteins were isolated from fibroblasts and analyzed by Western blot. Scramble shRNA was used as negative control. (D) Knockdown of PYCR1 (shPYCR1 #3, 2MOI) in skin fibroblast modulates the expression of extracellular space genes. (E) Evaluation of the overexpression of PYCR1 in human fibroblasts infected with 2, 5, and 30 MOI of recombinant PYCR1 lentiviruses relative to the empty vector control. (F) Western blot analyses revealed that PYCR1 overexpression (30 MOI) induce POSTN expression in skin fibroblast cells. (G) PYCR1 overexpression in patient’s skin fibroblasts (P2) modulates expression of extracellular space genes. Data was expressed as means ± SD from three biological replicates. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
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