F.M. Cornelis

Few Smad proteins and many Smad-interacting proteins yield multiple functions and action modes in TGFβ/BMP signaling in vivo

Signaling by the many ligands of the TGFβ family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models.

The effect of forced exercise on knee joints in Dio2−/− mice: type II iodothyronine deiodinase-deficient mice are less prone to develop OA-like cartilage damage upon excessive mechanical stress

Objective To further explore deiodinase iodothyronine type 2 (DIO2) as a therapeutic target in osteoarthritis (OA) by studying the effects of forced mechanical loading on in vivo joint cartilage tissue homeostasis and the modulating effect herein of Dio2 deficiency. Methods Wild-type and C57BL/6-Dio2−/− -mice were subjected to a forced running regime for 1 h per day for 3 weeks. Severity of OA was assessed by histological scoring for cartilage damage and synovitis. Genome-wide gene expression was determined in knee cartilage by microarray analysis (Illumina MouseWG-6 v2). STRING-db analyses were applied to determine enrichment for specific pathways and to visualise protein–protein interactions. Results In total, 158 probes representing 147 unique genes showed significantly differential expression with a fold-change ≥1.5 upon forced exercise. Among these are genes known for their association with OA (eg, Mef2c, Egfr, Ctgf, Prg4 and Ctnnb1), supporting the use of forced running as an OA model in mice. Dio2-deficient mice showed significantly less cartilage damage and signs of synovitis. Gene expression response upon exercise between wild-type and knockout mice was significantly different for 29 genes. Conclusions Mice subjected to a running regime have significant increased cartilage damage and synovitis scores. Lack of Dio2 protected against cartilage damage in this model and was reflected in a specific gene expression profile, and either mark a favourable effect in the Dio2 knockout (eg, Gnas) or an unfavourable effect in wild-type cartilage homeostasis (eg, Hmbg2 and Calr). These data further support DIO2 activity as a therapeutic target in OA.

A broken heart: A stretch too far: An overview of mouse models with mutations in stretch-sensor components

With every heartbeat the heart must contract and relax. This seemingly trivial process critically needs tight control of contraction and relaxation phases, and extremely efficient coordination between these two phases to control blood flow and maintain cardiac homeostasis. To achieve this, specialized sensors are required to detect the inherent repeatedly changing environment and needs. One sensor is a stretch-sensor that monitors the filling of the ventricles. Its molecular identity and localization are only partly understood. Here we give a synopsis of the genetic models that leap into our understanding of stretch-sensors. We focus on the widely acknowledged sarcomeric sensor at the Z-disc and the costamere sensor at the sarcolemma. Recently, several novel components of both sensors were discovered. Given that these two sensors seem physically connected, it is likely that these two models are not mutually exclusive and might even communicate. We describe briefly how candidate and known proteins within these sensors receive and transduce mechanical signals in the cardiomyocyte that lead to changes in gene expression underlying homeostasis and its restoration in the heart. Emphasis is placed on the putative link between altered stretch-sensor function and heart failure observed in different genetic mouse models of stretch-sensor components.

DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

Osteoarthritis is the most prevalent and crippling joint disease, and lacks curative treatment, as the underlying molecular basis is unclear. Here, we show that DOT1L, an enzyme involved in histone methylation, is a master protector of cartilage health. Loss of DOT1L disrupts the molecular signature of healthy chondrocytes in vitro and causes osteoarthritis in mice. Mechanistically, the protective function of DOT1L is attributable to inhibition of Wnt signalling, a pathway that when hyper-activated can lead to joint disease. Unexpectedly, DOT1L suppresses Wnt signalling by inhibiting the activity of sirtuin-1 (SIRT1), an important regulator of gene transcription. Inhibition of SIRT1 protects against osteoarthritis triggered by loss of DOT1L activity. Modulating the DOT1L network might therefore be a therapeutic approach to protect the cartilage against osteoarthritis.

Antagonism of Nodal signaling by BMP/Smad5 prevents ectopic primitive streak formation in the mouse amnion.

The strength and spatiotemporal activity of Nodal signaling is tightly controlled in early implantation mouse embryos, including by autoregulation and feedback loops, and involves secreted and intracellular antagonists. These control mechanisms, which are established at the extra-embryonic/embryonic interfaces, are essential for anterior-posterior patterning of the epiblast and correct positioning of the primitive streak. Formation of an ectopic primitive streak, or streak expansion, has previously been reported in mutants lacking antagonists that target Nodal signaling. Here, we demonstrate that loss-of-function of a major bone morphogenetic protein (BMP) effector, Smad5, results in formation of an ectopic primitive streak-like structure in mutant amnion accompanied by ectopic Nodal expression. This suggests that BMP/Smad5 signaling contributes to negative regulation of Nodal. In cultured cells, we find that BMP-activated Smad5 antagonizes Nodal signaling by interfering with the Nodal-Smad2/4-Foxh1 autoregulatory pathway through the formation of an unusual BMP4-induced Smad complex containing Smad2 and Smad5. Quantitative expression analysis supports that ectopic Nodal expression in the Smad5 mutant amnion is induced by the Nodal autoregulatory loop and a slow positive-feedback loop. The latter involves BMP4 signaling and also induction of ectopic Wnt3. Ectopic activation of these Nodal feedback loops in the Smad5 mutant amnion results in the eventual formation of an ectopic primitive streak-like structure. We conclude that antagonism of Nodal signaling by BMP/Smad5 signaling prevents primitive streak formation in the amnion of normal mouse embryos.

ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone

ANP32A is a transcriptional regulator of ATM and provides protection against oxidative stress to prevent joint, brain, and bone disease. Oxidative stress and osteoarthritis Osteoarthritis is a common degenerative joint disorder that affects cartilage and bone. Cornelis et al. investigated the role of ANP32A, a protein involved in multiple cellular processes, in osteoarthritis. ANP32A was decreased in osteoarthritic human and mouse tissue samples and also decreased with aging. The authors found that ANP32A promoted transcription of ATM and regulated reactive oxygen species in cartilage. Antioxidant therapy protected Anp32a-deficient mice from developing osteoarthritis and osteopenia and also rescued neurological defects caused by lack of ATM and increased oxidative stress. These results suggest that ANP32A could be a therapeutic target for correcting imbalanced reactive oxygen species and antioxidants. Osteoarthritis is the most common joint disorder with increasing global prevalence due to aging of the population. Current therapy is limited to symptom relief, yet there is no cure. Its multifactorial etiology includes oxidative stress and overproduction of reactive oxygen species, but the regulation of these processes in the joint is insufficiently understood. We report that ANP32A protects the cartilage against oxidative stress, preventing osteoarthritis development and disease progression. ANP32A is down-regulated in human and mouse osteoarthritic cartilage. Microarray profiling revealed that ANP32A protects the joint by promoting the expression of ATM, a key regulator of the cellular oxidative defense. Antioxidant treatment reduced the severity of osteoarthritis, osteopenia, and cerebellar ataxia features in Anp32a-deficient mice, revealing that the ANP32A/ATM axis discovered in cartilage is also present in brain and bone. Our findings indicate that modulating ANP32A signaling could help manage oxidative stress in cartilage, brain, and bone with therapeutic implications for osteoarthritis, neurological disease, and osteoporosis.

Aberrant Calreticulin Expression in Articular Cartilage of Dio2 Deficient Mice

Objective To identify intrinsic differences in cartilage gene expression profiles between wild-type- and Dio2-/--mice, as a mechanism to investigate factors that contribute to prolonged healthy tissue homeostasis. Methods Previously generated microarray-data (Illumina MouseWG-6 v2) of knee cartilage of wild-type and Dio2 -/- -mice were re-analyzed to identify differential expressed genes independent of mechanical loading conditions by forced treadmill-running. RT-qPCR and western blot analyses of overexpression and knockdown of Calr in mouse chondro-progenitor cells (ATDC5) were applied to assess the direct effect of differential Calr expression on cartilage deposition. Results Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > |1,5|) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044). Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition. Conclusion We here demonstrate that the beneficial homeostatic state of articular cartilage in Dio2-/- mice is accompanied with significant lower expression of Calr. Functional analyses further showed that upregulation of Calr expression could act as an initiator of cartilage destruction. The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

A8.04 The histone methyltransferase dot1l is essential for growth and homeostasis of the articular cartilage

Background and objectives DOT1L is the only known H3K79 histone methyltransferase. Genome-wide association and functional studies identified the DOT1L gene to be associated with cartilage thickness and hip osteoarthritis (OA) and showed an interaction of DOT1L with canonical Wnt signalling. Variations in the DOT1L gene are also associated with human height. These findings and our earlier in vitro insights prompted us to investigate the impact of DOT1L loss of function in vivo. Methods We generated a conditional cartilage-specific knockout (KO) model of DOT1L by crossing Dot1lfl/fl mice with Col2-Cre+/- mice. The deletion of exon2 in the Dot1l gene in cartilage was confirmed by PCR. We performed skeletal staining to identify skeletal abnormalities and growth defects, and histology of the growth plate. To study if the absence of DOT1L activity causes the development of early OA, we injected the chemical DOT1L inhibitor EPZ-5676 intra-articularly in the right knee of 8-week-old wild-type C57Bl/6 mice. Severity of disease was determined by histological scores on sections throughout the knee. Both cartilage damage and synovial hyperplasia were assessed based on OARSI guidelines. Based in our in vitro observations, we analysed the effect of DOT1L loss of function on specific Wnt target genes in either DOT1L cartilage-specific KO mice or in mice injected with EPZ-5676, by immunohistochemistry. Results A growth defect resulting in short stature became quickly apparent and resulted in mortality from the age of 4-weeks onwards in Dot1l cartilage-specific KO mice. These DOT1L deficient mice exhibited changes in the growth plates with a reduced and disorganised proliferative and pre-hypertrophic zone. In mice injected with EPZ-5676, histology severity scores were significantly increased over time compared to control treatment groups. We confirmed the regulatory role of DOT1L on canonical Wnt signalling in cartilage, since the absence of DOT1L activity in either cartilage specific KO mice or mice injected with DOT1L inhibitor resulted in increased protein levels of specific Wnt target genes. Conclusions Our findings support an essential role for DOT1L in growth and cartilage homeostasis as a key regulator of canonical WNT signalling in the joint.