Cellular stress modulates severity of the inflammatory response in lungs via cell surface BiP.

Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we observed increased levels of cell surface BiP (cs-BiP) in leukocytes during inflammation. This corresponds with a higher number of neutrophiles, which show naturally high levels of cs-BiP, whereas alveolar macrophages show a higher than usual exposure of BiP in their cell surface. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.

4-PBA Treatment Improves Bone Phenotypes in the Aga2 Mouse Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetically heterogenous disorder most often due to heterozygosity for mutations in the type I procollagen genes, COL1A1 or COL1A2. The disorder is characterized by bone fragility leading to increased fracture incidence and long-bone deformities. Although multiple mechanisms underlie OI, endoplasmic reticulum (ER) stress as a cellular response to defective collagen trafficking is emerging as a contributor to OI pathogenesis. Herein, we used 4-phenylbutiric acid (4-PBA), an established chemical chaperone, to determine if treatment of Aga2+/- mice, a model for moderately severe OI due to a Col1a1 structural mutation, could attenuate the phenotype. In vitro, Aga2+/- osteoblasts show increased protein kinase RNA-like endoplasmic reticulum kinase (PERK) activation protein levels, which improved upon treatment with 4-PBA. The in vivo data demonstrate that a postweaning 5-week 4-PBA treatment increased total body length and weight, decreased fracture incidence, increased femoral bone volume fraction (BV/TV), and increased cortical thickness. These findings were associated with in vivo evidence of decreased bone-derived protein levels of the ER stress markers binding immunoglobulin protein (BiP), CCAAT/-enhancer-binding protein homologous protein (CHOP), and activating transcription factor 4 (ATF4) as well as increased levels of the autophagosome marker light chain 3A/B (LC3A/B). Genetic ablation of CHOP in Aga2+/- mice resulted in increased severity of the Aga2+/- phenotype, suggesting that the reduction in CHOP observed in vitro after treatment is a consequence rather than a cause of reduced ER stress. These findings suggest the potential use of chemical chaperones as an adjunct treatment for forms of OI associated with ER stress. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Skeletal diseases caused by mutations in PTH1R show aberrant differentiation of skeletal progenitors due to dysregulation of DEPTOR.

Alterations in the balance between skeletogenesis and adipogenesis is a pathogenic feature in multiple skeletal disorders. Clinically, enhanced bone marrow adiposity in bones impairs mobility and increases fracture risk, reducing the quality of life of patients. The molecular mechanism that underlies the balance between skeletogenesis and adipogenesis is not completely understood but alterations in skeletal progenitor cells' differentiation pathway plays a key role. We recently demonstrated that parathyroid hormone (PTH)/PTH-related peptide (PTHrP) control the levels of DEPTOR, an inhibitor of the mechanistic target of rapamycin (mTOR), and that DEPTOR levels are altered in different skeletal diseases. Here, we show that mutations in the PTH receptor-1 (PTH1R) alter the differentiation of skeletal progenitors in two different skeletal genetic disorders and lead to accumulation of fat or cartilage in bones. Mechanistically, DEPTOR controls the subcellular localization of TAZ (transcriptional co-activator with a PDZ-binding domain), a transcriptional regulator that governs skeletal stem cells differentiation into either bone and fat. We show that DEPTOR regulation of TAZ localization is achieved through the control of Dishevelled2 (DVL2) phosphorylation. Depending on nutrient availability, DEPTOR directly interacts with PTH1R to regulate PTH/PTHrP signaling or it forms a complex with TAZ, to prevent its translocation to the nucleus and therefore inhibit its transcriptional activity. Our data point DEPTOR as a key molecule in skeletal progenitor differentiation; its dysregulation under pathologic conditions results in aberrant bone/fat balance.

DEPTOR in Skeletal Development and Diseases.

Discovered in 2009, the DEP-domain containing mTOR-interacting protein, DEPTOR, is a known regulator of the mechanistic target of rapamycin (mTOR), an evolutionarily conserved kinase that regulates diverse cellular processes in response to environmental stimuli. DEPTOR was originally identified as a negative regulator of mTOR complexes 1 (mTORC1) and 2 (mTORC2). However, recent discoveries have started to unravel the roles of DEPTOR in mTOR-independent responses. In the past few years, mTOR emerged as an important regulator of skeletal development, growth, and homeostasis; the dysregulation of its activity contributes to the development of several skeletal diseases, both chronic and genetic. Even more recently, several groups have reported on the relevance of DEPTOR in skeletal biology through its action on mTOR-dependent and mTOR-independent pathways. In this review, we summarize the current understanding of DEPTOR in skeletal development and disease.

An RNA aptamer restores defective bone growth in FGFR3-related skeletal dysplasia in mice.

Achondroplasia is the most prevalent genetic form of dwarfism in humans and is caused by activating mutations in FGFR3 tyrosine kinase. The clinical need for a safe and effective inhibitor of FGFR3 is unmet, leaving achondroplasia currently incurable. Here, we evaluated RBM-007, an RNA aptamer previously developed to neutralize the FGFR3 ligand FGF2, for its activity against FGFR3. In cultured rat chondrocytes or mouse embryonal tibia organ culture, RBM-007 rescued the proliferation arrest, degradation of cartilaginous extracellular matrix, premature senescence, and impaired hypertrophic differentiation induced by FGFR3 signaling. In cartilage xenografts derived from induced pluripotent stem cells from individuals with achondroplasia, RBM-007 rescued impaired chondrocyte differentiation and maturation. When delivered by subcutaneous injection, RBM-007 restored defective skeletal growth in a mouse model of achondroplasia. We thus demonstrate a ligand-trap concept of targeting the cartilage FGFR3 and delineate a potential therapeutic approach for achondroplasia and other FGFR3-related skeletal dysplasias.

Biallelic mutations in LAMA5 disrupts a skeletal noncanonical focal adhesion pathway and produces a distinct bent bone dysplasia.

BACKGROUND: Beyond its structural role in the skeleton, the extracellular matrix (ECM), particularly basement membrane proteins, facilitates communication with intracellular signaling pathways and cell to cell interactions to control differentiation, proliferation, migration and survival. Alterations in extracellular proteins cause a number of skeletal disorders, yet the consequences of an abnormal ECM on cellular communication remains less well understood METHODS: Clinical and radiographic examinations defined the phenotype in this unappreciated bent bone skeletal disorder. Exome analysis identified the genetic alteration, confirmed by Sanger sequencing. Quantitative PCR, western blot analyses, immunohistochemistry, luciferase assay for WNT signaling were employed to determine RNA, proteins levels and localization, and dissect out the underlying cell signaling abnormalities.  Migration and wound healing assays examined cell migration properties. FINDINGS: This bent bone dysplasia resulted from biallelic mutations in LAMA5, the gene encoding the alpha-5 laminin basement membrane protein. This finding uncovered a mechanism of disease driven by ECM-cell interactions between alpha-5-containing laminins, and integrin-mediated focal adhesion signaling, particularly in cartilage. Loss of LAMA5 altered ß1 integrin signaling through the non-canonical kinase PYK2 and the skeletal enriched SRC kinase, FYN. Loss of LAMA5 negatively impacted the actin cytoskeleton, vinculin localization, and WNT signaling. INTERPRETATION: This newly described mechanism revealed a LAMA5-ß1 Integrin-PYK2-FYN focal adhesion complex that regulates skeletogenesis, impacted WNT signaling and, when dysregulated, produced a distinct skeletal disorder. FUNDING: Supported by NIH awards R01 AR066124, R01 DE019567, R01 HD070394, and U54HG006493, and Czech Republic grants INTER-ACTION LTAUSA19030, V18-08-00567 and GA19-20123S.

Dominant-negative SOX9 mutations in campomelic dysplasia.

Campomelic dysplasia (CD) is an autosomal dominant, perinatal lethal skeletal dysplasia characterized by a small chest and short long bones with bowing of the lower extremities. CD is the result of heterozygosity for mutations in the gene encoding the chondrogenesis master regulator, SOX9. Loss-of-function mutations have been identified in most CD cases so it has been assumed that the disease results from haploinsufficiency for SOX9. Here, we identified distal truncating SOX9 mutations in four unrelated CD cases. The mutations all leave the dimerization and DNA-binding domains intact and cultured chondrocytes from three of the four cases synthesized truncated SOX9. Relative to CD resulting from haploinsufficiency, there was decreased transactivation activity toward a major transcriptional target, COL2A1, consistent with the mutations exerting a dominant-negative effect. For one of the cases, the phenotypic consequence was a very severe form of CD, with a pronounced effect on vertebral and limb development. The data identify a novel molecular mechanism of disease in CD in which the truncated protein leads to a distinct and more significant effect on SOX9 function.

The PTH/PTHrP-SIK3 pathway affects skeletogenesis through altered mTOR signaling.

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

NRP1 haploinsufficiency predisposes to the development of Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect. It involves anatomical abnormalities that change the normal flow of blood through the heart resulting in low oxygenation. Although not all of the underlying causes of TOF are completely understood, the disease has been associated with varying genetic etiologies including chromosomal abnormalities and Mendelian disorders, but can also occur as an isolated defect. In this report, we describe a familial case of TOF associated with a 1.8 Mb deletion of chromosome 10p11. Among the three genes in the region one is Neuropilin1 (NRP1), a membrane co-receptor of VEGF that modulates vasculogenesis. Hemizygous levels of NRP1 resulted in a reduced expression at the transcriptional and protein levels in patient-derived cells. Reduction of NRP1 also lead to decreased function of its activity as a co-receptor in intermolecular VEGF signaling. These findings support that diminished levels of NRP1 contribute to the development of TOF, likely through its function in mediating VEGF signal and vasculogenesis.

Corrigendum: Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle.

[This corrects the article on p. 889 in vol. 8, PMID: 28611805.].

Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle.

The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.

Comprehensive Tissue-Specific Transcriptome Analysis Reveals Distinct Regulatory Programs during Early Tomato Fruit Development.

Fruit formation and early development involve a range of physiological and morphological transformations of the various constituent tissues of the ovary. These developmental changes vary considerably according to tissue type, but molecular analyses at an organ-wide level inevitably obscure many tissue-specific phenomena. We used laser-capture microdissection coupled to high-throughput RNA sequencing to analyze the transcriptome of ovaries and fruit tissues of the wild tomato species Solanum pimpinellifolium. This laser-capture microdissection-high-throughput RNA sequencing approach allowed quantitative global profiling of gene expression at previously unobtainable levels of spatial resolution, revealing numerous contrasting transcriptome profiles and uncovering rare and cell type-specific transcripts. Coexpressed gene clusters linked specific tissues and stages to major transcriptional changes underlying the ovary-to-fruit transition and provided evidence of regulatory modules related to cell division, photosynthesis, and auxin transport in internal fruit tissues, together with parallel specialization of the pericarp transcriptome in stress responses and secondary metabolism. Analysis of transcription factor expression and regulatory motifs indicated putative gene regulatory modules that may regulate the development of different tissues and hormonal processes. Major alterations in the expression of hormone metabolic and signaling components illustrate the complex hormonal control underpinning fruit formation, with intricate spatiotemporal variations suggesting separate regulatory programs.

Mechanisms regulating auxin action during fruit development.

Auxin controls many aspects of fruit development, including fruit set and growth, ripening and abscission. However, the mechanisms by which auxin regulates these processes are still poorly understood. While it is generally agreed that precise spatial and temporal control of auxin distribution and signaling are required for fruit development, the dynamics of auxin biosynthesis and the mechanisms for its transport to different fruit tissues are mostly unknown. Despite major advances in elucidating many aspects of auxin biology in vegetative tissues, until recently, the nature and importance of auxin metabolism, transport and signaling during fruit ontogeny remained obscure. In this review, we summarize recent research that has started to elucidate the molecular mechanisms by which auxin is produced and transported in the fruit and to unravel the complexity of auxin signaling during fruit development. We also discuss recent approaches used to reveal the genes and regulatory networks that mediate cell and tissue-specific control of auxin levels in the developing fruit.

Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB.

• We have reported previously that the gibberellin (GA) content in strawberry receptacle is high, peaking at specific stages, pointing to a role of this hormone in fruit development. In Arabidopsis, miR159 levels are dependent on GA concentration. This prompted us to investigate the role of two members of the miR159 family and their putative strawberry target gene, GAMYB, in relation to changes in GA content during the course of fruit development. • The highest expression level of the two Fa-MIR159 genes was in the fruit's receptacle tissue, with dramatic changes observed throughout development. The lowest levels of total mature miR159 (a and b) were observed during the white stage of receptacle development, which was concurrent with the highest expression of Fa-GAMYB. A functional interaction between miR159 and Fa-GAMYB has been demonstrated in receptacle tissue. • The application of bioactive GA (i.e. GA(3) ) to strawberry plants caused the down-regulated expression of Fa-MIR159a, but the expression of Fa-MIR159b was not affected significantly. Clear discrepancies between Fa-MIR159b and mature Fa-miR159b levels were indicative of post-transcriptional regulation of Fa-MIR159b gene expression. • We propose that Fa-miR159a and Fa-miR159b interact with Fa-GAMYB during the course of strawberry receptacle development, and that they act in a cooperative fashion to respond, in part, to changes in GA endogenous levels.

Gibberellin biosynthesis and signalling during development of the strawberry receptacle.

The enlargement of receptacle cells during strawberry (Fragaria × ananassa) fruit development is a critical factor determining fruit size, with the increase in cell expansion being one of the most important physiological processes regulated by the phytohormone gibberellin (GA). Here, we studied the role of GA during strawberry fruit development by analyzing the endogenous content of bioactive GAs and the expression of key components of GA signalling and metabolism. Bioactive GA(1) , GA(3) and GA(4) were monitored during fruit development, with the content of GA(4) being extremely high in the receptacle, peaking at the white stage of development. •Genes with high homology to genes encoding GA pathway components, including receptors (FaGID1(GIBBERELLIN-INSENSITIVE DWARF1)b and FaGID1c), DELLA (FaRGA(REPRESSOR OF GA) and FaGAI(GA-INSENSITIVE)), and enzymes involved in GA biosynthesis (FaGA3ox) and catabolism (FaGA2ox), were identified, and their expression in different tissues and developmental stages of strawberry fruit was studied in detail. The expression of all of these genes showed a stage-specific pattern during fruit development and was highest in the receptacle. FaGID1c bound GA in vitro, interacted with FaRGA in vitro and in vivo, and increased GA responses when ectopically expressed in Arabidopsis. This study thus reveals key elements of GA responses in strawberry and points to a critical role for GA in the development of the receptacle.

Generation and analysis of ESTs from strawberry (Fragaria xananassa) fruits and evaluation of their utility in genetic and molecular studies.

BACKGROUND: Cultivated strawberry is a hybrid octoploid species (Fragaria xananassa Duchesne ex. Rozier) whose fruit is highly appreciated due to its organoleptic properties and health benefits. Despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. Further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative Fragaria vesca. However, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. We have produced a collection of expressed sequence tags (ESTs) from different cDNA libraries prepared from different fruit parts and developmental stages. The collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. The study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith. RESULTS: We have sequenced over 4,500 ESTs from Fragaria xananassa, thus doubling the number of ESTs available in the GenBank of this species. We then assembled this information together with that available from F. xananassa resulting a total of 7,096 unigenes. The identification of SSRs and SNPs in many of the ESTs allowed their conversion into functional molecular markers. The availability of libraries prepared from green growing fruits has allowed the cloning of cDNAs encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. In addition, the sequence information generated in the project, jointly with previous information on sequences from both F. xananassa and F. vesca, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. Comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative. CONCLUSIONS: The present study generates essential knowledge and molecular tools that will be useful in improving investigations at the molecular level in cultivated strawberry (F. xananassa). This knowledge is likely to provide useful resources in the ongoing breeding programs. The sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in QTL analysis. Massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes.

Modification of plant hormone levels and signaling as a tool in plant biotechnology.

Plant hormones are signal molecules, present in trace quantities, that act as major regulators of plant growth and development. They are involved in a wide range of processes such as elongation, flowering, root formation and vascular differentiation. For many years, agriculturists have applied hormones to their crops to either increase the yield, or improve the quality of the commercial product. Nowadays, the knowledge of hormone biosynthesis, degradation and signaling pathways has allowed the utilization of biotechnological tools to further improve the main agricultural crops. Natural or artificial mutants, with impaired functioning of the corresponding genes, have been adopted because of their superior phenotype in specific agricultural traits. In addition, transgenic plants have been generated to regulate internal hormone levels, or their signaling pathways, resulting in some crops that have revolutionized agriculture.