Aimy Sebastian

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner ☆

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1–/– mice lack any obvious limb or skeletal defects, Sost–/– mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost–/–; Sostdc1–/– mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost–/– and Sost–/–; Sostdc1–/– mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.

Global molecular changes in a tibial compression induced ACL rupture model of post-traumatic osteoarthritis.

Joint injury causes post‐traumatic osteoarthritis (PTOA). About ∼50% of patients rupturing their anterior cruciate ligament (ACL) will develop PTOA within 1–2 decades of the injury, yet the mechanisms responsible for the development of PTOA after joint injury are not well understood. In this study, we examined whole joint gene expression by RNA sequencing (RNAseq) at 1 day, 1‐, 6‐, and 12 weeks post injury, in a non‐invasive tibial compression (TC) overload mouse model of PTOA that mimics ACL rupture in humans. We identified 1446 genes differentially regulated between injured and contralateral joints. This includes known regulators of osteoarthritis such as MMP3, FN1, and COMP, and several new genes including Suco, Sorcs2, and Medag. We also identified 18 long noncoding RNAs that are differentially expressed in the injured joints. By comparing our data to gene expression data generated using the surgical destabilization of the medial meniscus (DMM) PTOA model, we identified several common genes and shared mechanisms. Our study highlights several differences between these two models and suggests that the TC model may be a more rapidly progressing model of PTOA. This study provides the first account of gene expression changes associated with PTOA development and progression in a TC model.

Transcriptional control of Sost in bone

Sclerostin is an osteocyte derived negative regulator of bone formation. A highly specific expression pattern and the exclusive bone phenotype have made Sclerostin an attractive target for therapeutic intervention in treating metabolic bone diseases such as osteoporosis and in facilitating fracture repair. Understanding the molecular mechanisms that regulate Sclerostin transcription is of great interest as it may unveil new avenues for therapeutic approaches. Such studies may also elucidate how various signaling pathways intersect to modulate bone metabolism. Here we review the current understanding of the upstream molecular mechanisms that regulate Sost/SOST transcription, in bone.

SOST Inhibits Prostate Cancer Invasion

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings.

Cancer–Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer

Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer–bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (SostKO) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-SostKO osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with SostKO osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.

SOST/Sclerostin Improves Posttraumatic Osteoarthritis and Inhibits MMP2/3 Expression After Injury: SOST OVEREXPRESSION IMPROVES PTOA OUTCOMES

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post‐injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG) and knockout (Sost‐/‐) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post‐injury compared with wild‐type (WT) controls and Sost‐/‐. In addition, SOSTTG built ∼50% and ∼65% less osteophyte volume than WT and Sost‐/‐, respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ∼2‐fold less MMP activation than WT or Sost‐/‐, and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra‐articular administration of recombinant Sost protein, immediately post‐injury, also significantly decreased MMP activity levels relative to PBS‐treated controls, and Sost activation in response to injury was TNFα and NF‐κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation.

Genetics of Sost/SOST in sclerosteosis and van Buchem disease animal models

Sclerosteosis and van Buchem disease (VBD) are two rare autosomal recessive disorders that results from osteoblast hyperactivity, in which progressive bone overgrowth leads to very dense bones, distortion of the face, and entrapment of cranial nerves. Sclerosteosis is caused by loss-of-function mutations in the SOST gene which encodes a secreted glycoprotein, sclerostin. VBD is caused by a noncoding deletion that removes a SOST-specific regulatory element in bone. In bone, SOST is expressed predominantly by osteocytes and sclerostin suppresses bone formation by inhibiting the canonical Wnt signaling pathway. Here we describe how human genetics studies in sclerosteosis and VBD patients, in combination with the generation of transgenic and knockout mice, has led to a better understanding of the role of sclerostin in bone metabolism.

Global gene expression analysis identifies Mef2c as a potential player in Wnt16-mediated transcriptional regulation

Wnt16 is a major Wnt ligand involved in the regulation of postnatal bone homeostasis. Previous studies have shown that Wnt16 promotes bone formation and inhibits bone resorption, suggesting that this molecule could be targeted for therapeutic interventions to treat bone thinning disorders such as osteoporosis. However, the molecular mechanisms by which Wnt16 regulates bone metabolism is not yet fully understood. To better understand the molecular mechanisms by which Wnt16 promotes bone formation and to identify the target genes regulated by Wnt16 in osteoblasts, we treated calvarial osteoblasts purified from C57Bl/6 mice with recombinant Wnt16 and profiled the gene expression changes by RNA-seq at 24 h post-treatment. We also compared gene expression profiles of Wnt16-treated osteoblasts to canonical Wnt3a- and non-canonical Wnt5a-treated osteoblasts. This study identified 576 genes differentially expressed in Wnt16-treated osteoblasts compared to sham-treated controls; these included several members of Wnt pathway (Wnt2b, Wnt7b, Wnt11, Axin2, Sfrp2, Sfrp4, Fzd5 etc.) and TGF-β/BMP signaling pathway (Bmp7, Inhba, Inhbb, Tgfb2 etc.). Wnt16 also regulated a large number of genes with known bone phenotypes. We also found that about 37% (215/576) of the Wnt16 targets overlapped with Wnt3a targets and ~15% (86/576) overlapped with Wnt5a targets, suggesting that Wnt16 activates both canonical and non-canonical Wnt signaling targets in osteoblasts. Transcription factor binding motif enrichment analysis in the promoter regions of Wnt16 targets identified noncanonical Wnt/JNK pathway activated transcription factors Fosl2 and Fosl1 as two of the most significantly enriched transcription factors associated with genes activated by Wnt16 while Mef2c was the most significantly enriched transcription factor associated with genes repressed by Wnt16. We also found that a large number of Mef2c targets overlapped with genes down-regulated by Wnt16 and Mef2c itself was transcriptionally repressed by Wnt16 suggesting that Mef2c plays a role in Wnt16-mediated transcriptional regulation.

Wnt co-receptors Lrp5 and Lrp6 differentially mediate Wnt3a signaling in osteoblasts

Wnt3a is a major regulator of bone metabolism however, very few of its target genes are known in bone. Wnt3a preferentially signals through transmembrane receptors Frizzled and co-receptors Lrp5/6 to activate the canonical signaling pathway. Previous studies have shown that the canonical Wnt co-receptors Lrp5 and Lrp6 also play an essential role in normal postnatal bone homeostasis, yet, very little is known about specific contributions by these co-receptors in Wnt3a-dependent signaling. We used high-throughput sequencing technology to identify target genes regulated by Wnt3a in osteoblasts and to elucidate the role of Lrp5 and Lrp6 in mediating Wnt3a signaling. Our study identified 782 genes regulated by Wnt3a in primary calvarial osteoblasts. Wnt3a up-regulated the expression of several key regulators of osteoblast proliferation/ early stages of differentiation while inhibiting genes expressed in later stages of osteoblastogenesis. We also found that Lrp6 is the key mediator of Wnt3a signaling in osteoblasts and Lrp5 played a less significant role in mediating Wnt3a signaling.

Comparative Transcriptomics Identifies Novel Genes and Pathways Involved in Post-Traumatic Osteoarthritis Development and Progression

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.