Osteoblasts orchestrate bone formation by secreting dense, highly cross-linked type I collagen and other proteins involved in osteogenesis. Mutations in Col1α1, Col1α2, or collagen biogenesis factors lead to the human genetic disease, osteogenesis imperfecta (OI). Herein, we show that the TENT5A gene, whose mutation is responsible for poorly characterized type XVIII OI, encodes an active cytoplasmic poly(A) polymerase regulating osteogenesis. TENT5A is induced during osteoblast differentiation and TENT5A KO osteoblasts are defective in mineralization. The TENT5A KO mouse recapitulates OI disease symptoms such as bone fragility and hypomineralization. Direct RNA sequencing revealed that TENT5A polyadenylates and increases expression of Col1α1 and Col1α2 RNAs, as well as those of other genes mutated in OI, resulting in lower production and improper folding of collagen chains. Thus, we have identified the specific pathomechanism of XVIII OI and report for the first time a biologically relevant post-transcriptional regulator of collagen production. We further postulate that TENT5A, possibly together with its paralogue TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization.

Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.

IntroductionAutoinflammatory diseases are characterized by dysregulation of innate immune system leading to spontaneous sterile inflammation. One of the well-established animal models of this group of disorders is the mouse strain Pstpip2cmo. In this strain, the loss of adaptor protein PSTPIP2 leads to the autoinflammatory disease chronic multifocal osteomyelitis. It is manifested by sterile inflammation of the bones and surrounding soft tissues of the hind limbs and tail. The disease development is propelled by elevated production of IL-1β and reactive oxygen species by neutrophil granulocytes. However, the molecular mechanisms linking PSTPIP2 and these pathways have not been established. Candidate proteins potentially involved in these mechanisms include PSTPIP2 binding partners, PEST family phosphatases (PEST-PTPs) and phosphoinositide phosphatase SHIP1.MethodsTo address the role of these proteins in PSTPIP2-mediated control of inflammation, we have generated mouse strains in which PEST-PTP or SHIP1 binding sites in PSTPIP2 have been disrupted. In these mouse strains, we followed disease symptoms and various inflammation markers.ResultsOur data show that mutation of the PEST-PTP binding site causes symptomatic disease, whereas mice lacking the SHIP1 interaction site remain asymptomatic. Importantly, both binding partners of PSTPIP2 contribute equally to the control of IL-1β production, while PEST-PTPs have a dominant role in the regulation of reactive oxygen species. In addition, the interaction of PEST-PTPs with PSTPIP2 regulates the production of the chemokine CXCL2 by neutrophils. Its secretion likely creates a positive feedback loop that drives neutrophil recruitment to the affected tissues.ConclusionsWe demonstrate that PSTPIP2-bound PEST-PTPs and SHIP1 together control the IL-1β pathway. In addition, PEST-PTPs have unique roles in the control of reactive oxygen species and chemokine production, which in the absence of PEST-PTP binding to PSTPIP2 shift the balance towards symptomatic disease.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system leading to spontaneous inflammation. Pstpip2 cmo mouse strain is a well-characterized model of this class of disorders. Due to the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1β by neutrophil granulocytes. Here we show that in addition to IL-1β, PSTPIP2 also negatively regulates ROS generation by neutrophil NADPH oxidase. Pstpip2 cmo neutrophils display highly elevated ROS production in response to a range of stimuli. Inactivation of NADPH oxidase in Pstpip2 cmo mice did not affect IL-1β levels and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2 cmo mice.

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