Investigation of β-Catenin-Mediated Regulation of IGFBP-6 and the Roles of IGFBP-6 and IGF-II in Dupuytren\u27s Disease

Dupuytren’s Disease (DD) is a benign and heritable connective tissue fibrosis that affects the palmar fascia and typically results in permanent finger contracture(s). Similar to other fibroses, DD is characterized by increased fibroblast proliferation, myofibroblast differentiation and excess collagen deposition. Currently, there are no truly effective treatment options for connective tissue fibroses. Increased levels of β-catenin, an intracellular trans-regulator of gene transcription, have been previously reported in DD. Genes that are associated with, and therefore potentially transcriptionally regulated by, β-catenin during DD development were identified in this thesis. One of these gene targets, IGFBP6, was shown to consistently be associated with β-catenin in fibroblasts derived from phenotypically normal palmar fascia (PF cells) but not fibroblasts derived from diseased tissues (DD cells). β-catenin association with the IGFBP6 promoter in these cells was directly correlated with IGFBP6 expression levels and with the secretion of its protein product, Insulin-like Growth Factor Binding Protein-6 (IGFBP-6). In addition, 1 438 unique genes were shown to associate with β-catenin in DD cells but not PF cells derived from the same patients. The functional consequences of IGFBP-6 repression, and the increased availability of its primary ligand, IGF-II were also elucidated. Exogenous addition of IGFBP-6 inhibited the proliferation of DD and control fibroblasts, and attenuated IGF-II induced contraction of DD cells. IGF-II stimulated the proliferation of normal fibroblasts but not fibroblasts derived from patients with DD. The gene encoding IGF-II, IGF2, was found to be up regulated in DD cells, and potential mechanisms facilitating IGF2 overexpression were investigated. Loss of imprinted expression of IGF2 was detected in a subset of patients and a corresponding loss of H19 expression, a non-coding RNA that is reciprocally expressed relative to IGF2, was observed. Aberrant IGF2 promoter usage was also identified in a subset of DD and PF cells. In combination, these disease-associated changes may explain the increased IGF2 expression in DD. Identification of novel genes targets of β-catenin and the factors that regulate the expression of IGFBP6 or IGF2 during the development of this debilitating fibrosis may allow us to identify novel therapeutic targets

IGF2 expression and β-catenin levels are increased in Frozen Shoulder Syndrome

Purpose: Frozen Shoulder Syndrome is a fibrosis of the shoulder joint capsule that is clinically associated with Dupuytren’s disease, a fibrosis of the palmar fascia. Little is known about any commonalities in the pathophysiology of these connective tissue fibroses. β-catenin, a protein that transactivates gene expression, and levels of IGF2 mRNA, encoding insulin-like growth factor-II, are elevated in Dupuytren’s disease. The aim of this study was to determine if correlating changes in β-catenin levels and IGF2 expression are evident in Frozen Shoulder Syndrome. Methods: Tissue from patients with Frozen Shoulder Syndrome and rotator cuff tear were obtained during shoulder arthroscopies. Total protein extracts were prepared from tissue aliquots and β-catenin immunoreactivity was assessed by Western immunoblotting. In parallel, primary fibroblasts were derived from these tissues and assessed for IGF2 expression by quantitative PCR. Results: β-catenin levels were significantly increased in Frozen Shoulder Syndrome relative to rotator cuff tear when assessed by Western immunoblotting analyses. IGF2 mRNA levels were significantly increased in primary fibroblasts derived from frozen shoulder syndrome tissues relative to fibroblasts derived from rotator cuff tissues. Conclusions: As in Dupuytren’s disease, β-catenin levels and IGF2 expression are elevated in Frozen Shoulder Syndrome. These findings support the hypothesis that these connective tissue fibroses share a common pathophysiology

Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

Abstract Background Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder. Conclusions These data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.</p