Role of PHOSPHO1 in periodontal development and function

The tooth root and periodontal apparatus, including the acellular and cellular cementum, periodontal ligament (PDL), and alveolar bone, are critical for tooth function. Cementum and bone mineralization is regulated by factors including enzymes and extracellular matrix proteins that promote or inhibit hydroxyapatite crystal growth. Orphan Phosphatase 1 (Phospho1, PHOSPHO1) is a phosphatase expressed by chondrocytes, osteoblasts, and odontoblasts that functions in skeletal and dentin mineralization by initiating deposition of hydroxyapatite inside membrane-limited matrix vesicles. The role of PHOSPHO1 in periodontal formation remains unknown and we aimed to determine its functional importance in these tissues. We hypothesized that the enzyme would regulate proper mineralization of the periodontal apparatus. Spatiotemporal expression of PHOSPHO1 was mapped during periodontal development, and Phospho1(-/-) mice were analyzed using histology, immunohistochemistry, in situ hybridization, radiography, and micro–computed tomography. The Phospho1 gene and PHOSPHO1 protein were expressed by active alveolar bone osteoblasts and cementoblasts during cellular cementum formation. In Phospho1(-/-) mice, acellular cementum formation and mineralization were unaffected, whereas cellular cementum deposition increased although it displayed delayed mineralization and cementoid. Phospho1(-/-) mice featured disturbances in alveolar bone mineralization, shown by accumulation of unmineralized osteoid matrix and interglobular patterns of protein deposition. Parallel to other skeletal sites, deposition of mineral-regulating protein osteopontin (OPN) was increased in alveolar bone in Phospho1(-/-) mice. In contrast to the skeleton, genetic ablation of Spp1, the gene encoding OPN, did not ameliorate dentoalveolar defects in Phospho1(-/-) mice. Despite alveolar bone mineralization defects, periodontal attachment and function appeared undisturbed in Phospho1(-/-) mice, with normal PDL architecture and no evidence of bone loss over time. This study highlights the role of PHOSPHO1 in mineralization of alveolar bone and cellular cementum, further revealing that acellular cementum formation is not substantially regulated by PHOSPHO1 and likely does not rely on matrix vesicle–mediated initiation of mineralization

Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling

Molecular pathology and embryology of Hoxd13 associated limb malformations

Patienten mit erblich bedingter Synpolydactylie (SPD) entwickeln Skelettfehlbildungen, wie einen oder mehrere, zusätzliche Finger oder Zehen und deren Fusionierung. Eine Mutation in Hoxd13, die zur Verlängerung eines Polyalaninrepeats führt, verursacht SPD; wobei die Schwere des entstehenden Phänotyps mit der Länge der Expansion korreliert ist. Hoxd13 gehört zur Familie der hoch konservierten Hox-Gene; Transkriptionsfaktoren, die während der embryonalen Entwicklung eine essentielle Rolle in der Achsenbildung spielen. In dieser Arbeit wurde das spontan entstandene Mausmodell spdh (synpolydactyly homolog) untersucht, um den Pathomechanismus der Hoxd13-assoziierten SPD aufzuklären. Spdh Tiere tragen eine Verlängerung um sieben zusätzliche Alanine in Hoxd13 und weisen einen SPD ähnlichen Phänotyp mit zusätzlichen Fingern und Zehen, deren Fusionierung, sowie defiziente Gelenkentwicklung und einen Verknöcherungsdefekt auf. Transgene Tiere und verschiedene Verkreuzungsexperimente lieferten Hinweise darauf, dass die Mutation in Hoxd13 eine Kombination aus Funktionsverlust und negativem Funktionsgewinn nach sich zieht, zumal auch Tiere mit inaktiviertem Hoxd13 keine SPD entwickeln. Zum einen ist es mit dieser Arbeit gelungen, Anhaltspunkte dafür zu finden, dass in spdh Tieren das Perichondrium als wesentlicher Signalgeber während der Ossifikation verloren geht, die verzögerte Verknöcherung nicht endochondral verläuft, und es sich hierbei um eine homeotische Transformation der Mittelhandknochen zu Handwurzelknochen handelt. Diese Effekte kommen vermutlich durch den Einfluss von mutiertem Hoxd13 auf Hoxa13 zustande. Zum anderen wird der Pathomechanismus aufgeklärt, der zu zusätzlichen, fusionierten Kondensationen führt. Mangelhafte BMP- Aktivierung und falsch regulierte Ephrin-vermittelte Signalübertragung führen zu mangelnder Apoptose und unausreichender Zell-Zellabgrenzung. Dies könnte für die Fusionierungen der chondrogenen Anlagen verantwortlich gemacht werden. Ausschlaggebend für die zusätzlichen Kondensationen ist der gestörte Retinsäuresignalweg. Hier wird gezeigt, dass Hoxd13 wt direkt an den Raldh2 Promotor binden und diesen aktivieren kann. Raldh2 ist das einzige Enzym, das in der entstehenden Extremitätenknospe Retinsäure (RA) produziert, die wiederum im interdigitalen Mesenchym die Chondrogenese unterdrückt. In spdh Tieren wird zuwenig Raldh2 aktiviert, daher zuwenig RA produziert; die Chondrogenese im interdigitalen Mesenchym wird nicht ausreichend unterdrückt und die Zellen dort erhalten falsche Differenzierungssignale. Die ungenügende RA-Konzentration konnte substituiert werden und behandelte Tiere entwickelten in allen Fällen fünf Finger. So konnte ein erblich bedingter Entwicklungsdefekt behoben werden. Daher wird hier ein neuer Pathomechanismus für Polydactylie vorgeschlagen, der im Gegensatz zu den bisher bekannten Mechanismen kein Defekt in der Musterbildung ist, sondern dem ein Prozess der unkontrollierten Differenzierung zugrunde liegt.Patients with inherited synpolydactyly (SPD) show limb malformations characterized by one ore more additional digits and toes and fusions of those. A mutation within Hoxd13 comprising an expansion of a polyalanine repeat is the cause for SPD. The length of the expansion is correlated to the severity of the phenotype. Hoxd13 belongs to the hox-gene family, transcription factors which play a crucial role in axis formation during embryonic development. In this work the mutant mouse model spdh (synpolydactyly homolog) was analyzed to elucidate the underlying pathomechanism of Hoxd13 associated SPD. Spdh animals carry an expansion of seven additional alanines and exhibit an SPD like phenotype with additional digits and toes, fusions, abnormal joint formation and a severe ossification delay. Transgenic approaches as well as crossbreeding studies revealed that the mutation in Hoxd13 results in a combination of loss and gain of function, particularly because animals with inactivated Hoxd13 do not display an SPD phenotype. On the one hand this work provides evidence, that in spdh animals the perichondrium, as essential cell layer, for ossification, is absent. Ossification is delayed and does not take place in an endochondral manner. This results in a homeotic transformation of metacarpals to carpal bones. These effects are likely due to an influence of mutated Hoxd13 on Hoxa13. On the other hand the pathomechanism leading to additional condensations was elucidated. BMP- and Ephrin-signaling pathways were shown to be altered. This contributes to deficient differentiation and lacking apoptosis as well as insufficient border formation, between the cartilaginous and non-cartilaginous tissue. This contributes to the fusion of condensations. Crucial for additional condensations is the deregulation of the retinoic acid (RA) signaling pathway. Here we show that Hoxd13 wt is able to bind directly to and to activate the Raldh2 promoter. Raldh2 is the unique enzyme in the developing limb bud which produces RA, which in turn suppresses chondrogenesis in the interdigital mesenchyme. In spdh mutants less Raldh2 is activated, resulting in reduced RA levels in the limbs. Due to reduced RA levels chondrogenesis is not inhibited properly and cells obtain wrong differentiation signals in the interdigital mesenchyme. Insufficient RA levels were restored by oral administration of RA to pregnant mice. Treated animals sowed five digits in all cases. Thus, the SPD phenotype was partially rescued. In conclusion a new pathomechanism for polydactyly was characterized, which is, in contrast to known mechanisms, not a patterning defect like mirror duplications, but rather a result of irregular uncontrolled processes of differentiation within the interdigital mesenchyme

Skeletal muscle in MuRF1 null mice is not spared in low-gravity conditions, indicating atrophy proceeds by unique mechanisms in space

Microgravity exposure is associated with loss of muscle mass and strength. The E3 ubiquitin ligase MuRF1 plays an integral role in degrading the contractile apparatus of skeletal muscle; MuRF1 null (KO) mice have shown protection in ground-based models of muscle atrophy. In contrast, MuRF1 KO mice subjected to 21 days of microgravity on the International Space Station (ISS) were not protected from muscle atrophy. In a time course experiment microgravity-induced muscle loss on the ISS showed MuRF1 gene expression was not upregulated. A comparison of the soleus transcriptome profiles between spaceflight and a publicly available data set for hindlimb suspension, a claimed surrogate model of microgravity, showed only marginal commonalities between the models. These findings demonstrate spaceflight induced atrophy is unique, and that understanding of effects of space requires study situated beyond the Earth's mesosphere

Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs

Agonists of mouse STING (TMEM173) shrink and even cure solid tumor by activating innate immunity; human STING agonists are needed to test this therapeutic hypothesis in man. The endogenous STING agonist is 2′3′-cGAMP, a 2nd messenger that signals the presence of cytosolic dsDNA. We report activity-guided partial purification and identification of ENPP1 as the dominant 2′3′-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1-/- mice. We synthesized a hydrolysis-resistant bis-phosphothioate analog of 2′3′-cGAMP (2′3′-cGsAsMP) with similar affinity for human STING in vitro and 10 times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1-/- lung fibroblasts indicate that resistance to hydrolysis contributes significantly to its higher potency. 2′3′-cGsAsMP is therefore improved over natural 2′3′-cGAMP as a model agonist, and has potential as a vaccine adjuvant and cancer therapeutic

Homeobox genes d11–d13 and a13 control mouse autopod cortical bone and joint formation

The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11–/–Hoxd12–/–Hoxd13–/– triple-knockout mice and Hoxd13–/–Hoxa13+/– mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2

"3D, human renal proximal tubule (RPTEC-TERT1) organoids 'tubuloids' for translatable evaluation of nephrotoxins in high-throughput".

The importance of human cell-based in vitro tools to drug development that are robust, accurate, and predictive cannot be understated. There has been significant effort in recent years to develop such platforms, with increased interest in 3D models that can recapitulate key aspects of biology that 2D models might not be able to deliver. We describe the development of a 3D human cell-based in vitro assay for the investigation of nephrotoxicity, using RPTEC-TERT1 cells. These RPTEC-TERT1 proximal tubule organoids 'tubuloids' demonstrate marked differences in physiologically relevant morphology compared to 2D monolayer cells, increased sensitivity to nephrotoxins observable via secreted protein, and with a higher degree of similarity to native human kidney tissue. Finally, tubuloids incubated with nephrotoxins demonstrate altered Na+/K+-ATPase signal intensity, a potential avenue for a high-throughput, translatable nephrotoxicity assay

Mutant Hoxd13 induces extra digits in a mouse model of synpolydactyly directly and by decreasing retinoic acid synthesis

Individuals with the birth defect synpolydactyly (SPD) have 1 or more digit duplicated and 2 or more digits fused together. One form of SPD is caused by polyalanine expansions in homeobox d13 (Hoxd13). Here we have used the naturally occurring mouse mutant that has the same mutation, the SPD homolog (Spdh) allele, and a similar phenotype, to investigate the molecular pathogenesis of SPD. A transgenic approach and crossing experiments showed that the Spdh allele is a combination of loss and gain of function. Here we identify retinaldehyde dehydrogenase 2 (Raldh2), the rate-limiting enzyme for retinoic acid (RA) synthesis in the limb, as a direct Hoxd13 target and show decreased RA production in limbs from Spdh/Spdh mice. Intrauterine treatment with RA restored pentadactyly in Spdh/Spdh mice. We further show that RA and WT Hoxd13 suppress chondrogenesis in mesenchymal progenitor cells, whereas Hoxd13 encoded by Spdh promotes cartilage formation in primary cells isolated from Spdh/Spdh limbs, and that this was associated with increased expression of Sox6/9. Increased Sox9 expression and ectopic cartilage formation in the interdigital mesenchyme of limbs from Spdh/Spdh mice suggest uncontrolled differentiation of these cells into the chondrocytic lineage. Thus, we propose that mutated Hoxd13 causes polydactyly in SPD by inducing extraneous interdigital chondrogenesis, both directly and indirectly, via a reduction in RA levels