Cloning and chromosomal assignment of a widely expressed human receptor-like protein-tyrosine phosphatase

AbstractInsight into the regulation of the actions of the protein-tyrosine kinases will be greatly facilitated by the full characterization of the family of protein-tyrosine phosphatases. A search for novel phosphatases resulted in the isolation of a cDNA, termed HLPR, encoding a member of the family of human receptor-like protein-tyrosine phosphatases: its cDNA sequence predicts a protein of 793 amino acids (unglycosylated Mr, 87500) and includes a 121 residue extracellular domain, a single transmembrane segment, and two tandem intra-cytoplasmic catalytic domains. The HLPR gene is located on human chromosome 20, and the protein it encodes likely plays a fundamental role in the physiology of all cells as its expression appears to be ubiquitous

Surviving Endoplasmic Reticulum Stress Is Coupled to Altered Chondrocyte Differentiation and Function

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded α1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia

SOX9 Governs Differentiation Stage-Specific Gene Expression in Growth Plate Chondrocytes via Direct Concomitant Transactivation and Repression

Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9–GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between these opposing modes of transcriptional control by SOX9 may be mediated by cooperation with different partners such as GLI factors

Synergistic co-regulation and competition by a SOX9-GLI-FOXA phasic transcriptional network coordinate chondrocyte differentiation transitions

<div><p>The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a <u><b>G</b></u>rowth <u><b>P</b></u>late <u><b>D</b></u>ifferential <u><b>G</b></u>ene <u><b>E</b></u>xpression <u><b>L</b></u>ibrary (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. <i>De novo</i> motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as <i>Trps1</i>, <i>Sox9</i>, <i>Sox5</i>, <i>Sox6</i>, <i>Col2a1</i>, <i>Ptch1</i>, <i>Gli1</i> and <i>Gli2</i>. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.</p></div

Co-localization of SOX9 and FOXA2 in the growth plate.

<p>(A-C) <i>In vivo</i> expression patterns of SOX9 (A, red), FOXA2 (B, green) and co-localization (C) were shown on the cryosectioned growth plate (P10). Boxed regions of PCs (a-a"), PHCs (b-b") and HCs (c-c") were shown in higher magnification to demonstrate the differential expression and co-localization of SOX9 and FOXA2. (Bar = 100μm).</p

Predicted regulation by SOX9 and GLI1 cooperation.

<p>(A-C) SOX9, GLI1 (Generated from E11.5 developing limb) ChIP-seq signals and conservation score in the loci of <i>Cyr61</i>, <i>Trps1</i> and <i>Ptch1</i>. BS: binding site; BR: binding region. (D-I) SOX9, GLI1 ChIP-seq signals and conservation score in the loci of Hedgehog target genes (<i>Gli1</i> and <i>Gli2</i>) and Sox9 target genes (<i>Sox9</i>, <i>Col2a1</i>, <i>Sox5</i> and <i>Sox6</i>).</p

Global gene expression profiling in different chondrocyte populations.

<p>(A) 10-day-old mouse growth plates were cryosectioned and mRNA was extracted from the pooled samples of chondrocytes in the PZ, PHZ, UHZ and LHZ. Microarray data were then generated for the expression profiling of 21464 genes in each population of chondrocytes. (B) A total of 1891 genes showed differential expression patterns over the 4 zones with coefficient of standard deviation (CSD) of mRNA levels greater than 0.15. Four major distinct patterns of gene expression over the growth plates were identified in Heatmap by using <i>K</i>-Means Clustering. (C) Enriched biological processes representing the main functions of the co-expressed genes in each cluster by GO term analysis.</p

Competition between SOX9 and FOXA2 during chondrocyte differentiation.

<p>(A-D) Competition between SOX9 and FOXA2 on <i>Col2a1</i> intron 1 and <i>Col10a1</i> enhancer by luciferase assay in ATDC5. Enh: enhancer; Pro: promoter. The amount of SOX9 was kept constant and FOXA2 was increased and vice versa. (E) EMSA was performed using probes for the <i>Col2a1</i> intron I (Gel I and II) and <i>Col10a1</i> enhancer (Gel III and IV), and SOX9 and FOXA proteins at indicated concentrations. The sequences from <i>Col2a1</i> intron I and <i>Col10a1</i> enhancer used in the EMSAs are shown in the upper panel, and the SOX9/FOXA binding motifs are indicated by capital letters. **: p-value < 0.01; *: p-value < 0.05; NS: Non-significant.</p

Functional validation of SOX9 and GLI cooperation.

<p>(A) Relative expression levels of SOX9-GLI common target genes in <i>Sox9</i>^<i>+/+</i> and <i>Sox9</i>^<i>+/-</i> limb chondrocytes (E13.5). (B) Relative expression levels of SOX9-GLI common target genes in <i>Gli2</i>^<i>+/+</i> and <i>Gli2</i>^<i>-/-</i> limb chondrocytes (E13.5). <i>Gapdh</i> was used as the endogenous control. The error bar represents the standard deviation of the relative expression level of each gene in independent triplicate. (C) SOX9 activated the expression of <i>Ptch1</i> promoter in a dosage dependent manner in transiently transfected ATDC5 cells. (D-H) SOX9 increased GLI-dependent activation of <i>Ptch1</i>, <i>Gli1</i>, <i>Gli2</i>, <i>Sox9</i> and <i>Col2a1</i> regulatory elements in transiently transfected ATDC5 cells. Expression plasmids for <i>Sox9</i>, <i>Gli1</i>, <i>Gli2</i> and their combination were cotransfected as indicated below the bars. Equal amounts of expression plasmids were used for each well. **: p-value < 0.01; *: p-value < 0.05; NS: Non-significant.</p

Expression patterns of putative SOX9 target genes and SOX9 binding regions in their genomic loci.

<p>(A-I) Genes with SOX9 binding motifs located within 200bp from SOX9 ChIP-seq peaks (Generated from newborn rib chondrocytes) were selected for validation. <i>In vivo</i> expression patterns of potential SOX9 targets were validated by in situ hybridization: <i>Zbtb20</i> (A), <i>Wwp2</i> (B), <i>Ppa1</i> (D), <i>Bnip3</i> (E), <i>Slc8a3</i> (F), <i>Wnk4</i> (G) and <i>Col10a1</i> (H); or immunostaining: FOXP2 (C) and SOX9 (I), revealing similar expression trends with the microarray data as shown on the left side. The PZ, PHZ and HZ were separated by the white-dot lines. (Bar = 100μm). Predicted SOX9 binding sites (BS: binding site) and SOX9 ChIP-seq signals (BR: binding region) (Green) are shown under each gene.</p