Bone synthesis should depend on autophagy because over 10% of type I procollagen (PC1) - a heterotrimer of COL1A1 and COL1A2 chains and the precursor of the main bone matrix molecule - is misfolded and rerouted from osteoblast endoplasmic reticulum (ER) to lysosomes. However, osteoblast-specific macroautophagy knockouts in mice have produced only mild bone effects. To reconcile these observations, we compared how hypomorphic expression and a conditional knockout (cKO) of Atg5 - encoding a protein required for autophagosome formation - affected Col1a2G610C/+ versus wild-type Col1a2+/+ osteoblasts in vivo and in vitro. The Gly610-to-Cys substitution (G610C) in the triple helical region of the COL1A2/proα2(I) chain increases PC1 misfolding, causing its accumulation in the ER, cell stress, and osteoblast malfunction. Because autophagy reroutes misfolded PC1 from the ER to lysosomes, disruption of PC1 autophagy should significantly increase osteoblast malfunction and bone pathology in Col1a2G610C/+ mice. Nonetheless, the present study revealed only minor effects of the atg5 cKO on osteoblast function and bone formation in the Col1a2G610C/+ mice, like in Col1a2+/+ controls. The cKO did not reduce the autophagy flux of misfolded G610C or wild-type PC1 in primary osteoblast cultures, even though the LC3 and GABARAP lipidation and therefore autophagosome formation were disrupted. Live-cell imaging in atg5 cKO osteoblasts demonstrated that PC1 was efficiently delivered to lysosomes without LC3 via ER exit site (ERES) microautophagy. Taken together, these observations indicate that LC3- and GABARAP-independent ERES microautophagy is the primary pathway of misfolded procollagen degradation in osteoblasts both in culture and in vivo.Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; Baf: bafilomycin A1; BFA: brefeldin A; BGLAP/Ocn/osteocalcin: bone gamma-carboxyglutamate protein; COL1A1/proα1(I): collagen type I alpha 1 chain; COL1A2/proα2(I): collagen type I alpha 2 chain; cKO: conditional knockout; ER: endoplasmic reticulum; ERES: ER exit site; G610C mutation: COL1A2 p.Gly706Cys replacing Gly in position 610 from the start of the triple helix with Cys; GABARAP: GABA type A receptor-associated protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAR: mineral apposition rate; Ob: osteoblast; Oc: osteoclast; OI: osteogenesis imperfecta; PC1: procollagen type I, a heterotrimer of two COL1A1 and one COL1A2 chains, precursor of collagen type I; PDI: protein disulfide isomerase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SP7/osterix: Sp7 transcription factor; SQSTM1/p62: sequestosome 1; WT: wild type.

ABSTRACT Relevance of mineralized nodules in two‐dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone‐like cells, extracellular matrix (ECM), and mineral were all reported but so were non‐bone‐like ones. Many studies described seemingly bone‐like cell‐ECM structures based on similarity to few select bone features in vivo, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and in situ messenger RNA (mRNA) hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form bona fide bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial–temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell‐ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage‐like structure with chondrocyte‐to‐osteoblast transdifferentiation. The two nonmineralizing cell‐ECM structures may mimic periosteal cambium and pathological, nonmineralizing osteoid. Importantly, the most commonly used culture conditions (10mM β‐glycerophosphate) induce artificial mineralization of all cell‐ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of bona fide bone and simple means for distinguishing it from the other cell‐ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

HIV-Tat protein is released extracellularly in large amounts from HIV-infected cells and causes activation of lymphocytes, glial cells and neurotoxicity, even under antiretroviral therapy. Our atomic force microscopy (AFM) and mass spectrometry data showed that HIV-Tat can produce spontaneously shorter peptides, some of which are consistent with the protein's predicted inteins. We found that the HIV-Tat 32-62 piece is neurotoxic by itself. AFM showed, at 32 micrograms/ml, formation of micron-long HIV-Tat 32-62 fibrillar structures. These fibrils present branching and have a persistence length in the 80-90 nm range. With increasing concentration, the fibrils thicken and their branching angles decrease. With further increasing concentration, large irregularly shaped aggregates form in solution and, long fibers and sheet-like aggregates both extending over tens of microns form at air-liquid interface. Birefringence microscopy, FTIR and polarized Raman microspectroscopies of the aggregates suggest that the peptide has highly oriented side chains and backbone and contains 45% beta-sheet, consistent with the amyloid fiber structure. This is different than the full-length HIV-Tat, which is alpha-helical in bound state and in aggregates. Unlike other amyloids, the peptide aggregates have a distinct mechanism of irreversibility. Raman measurements showed that the free cysteines in aggregates form in time S-S bonds, which make the aggregates insoluble. The replacement of Cys34, Phe38 and Ile45 with Ala in the peptide chain led to reduced aggregation, no fibril formation and lower neurotoxicity. We identified microns-long Tat 32-62 fibers in cells expressing the peptide or the full-length Tat. Our model of the thinnest fibril involves a beta sheet backbone of the fibril to which small aggregates of various sizes are bound. These results suggest that HIV-Tat autocleavage makes possible the aggregation of the 32-62 fragment which leads to neurotoxicity through an amyloid type of mechanism.

Cartilage is composed of cells and an extracellular matrix, the latter being a composite of a collagen mesh interpenetrated by proteoglycans responsible for tissue osmotic swelling. The matrix composition and structure vary through the tissue depth. Mapping such variability requires tissue sectioning to gain access. The resulting surface roughness, and concomitant proteoglycan loss contribute to large uncertainties in elastic modulus estimates. To extract elasticity values for the bulk matrix which are not obfuscated by the indeterminate surface layer, we developed a novel experimental and data analysis methodology. We analyzed the surface roughness to optimize the probe size, and performed high-resolution (1 μm) elasticity mapping on thin (∼12 μm), epiphyseal newborn mouse cartilage sections cut parallel to the bone longitudinal axis or normal to the articular surface. Mild fixation prevented the major proteoglycan loss observed in unfixed specimens but not the stress release that resulted in thickness changes in the sectioned matrix. Our novel data analysis method introduces a virtual contact point as a fitting parameter for the Hertz model, to minimize the effects of surface roughness and corrects for the finite section thickness. Our estimates of cartilage elasticity converge with increasing indentation depth and, unlike previous data interpretations, are consistent with linearly elastic material. A high cell density that leaves narrow matrix septa between cells may cause the underestimation of elastic moduli, whereas fixation probably causes an overestimation. The proposed methodology has broader relevance to nano- and micro-indentation of soft materials with multiple length scales of organization and whenever surface effects (including roughness, electrostatics, van der Waals forces, etc.) become significant.

Cartilage matrix is composed of a dense collagen matrix and of the highly-charged glycoprotein, aggrecan that is responsible for tissue osmotic swelling. The interplay of the two is presumably critical for the cushioning of bones at the joints. The relationship between osmotic and mechanical properties and composition is poorly understood and mapping of the related parameters through tissue depth is complicated by several issues relating to the need to section the tissue to gain access. Tissue damage and intrinsic roughness at the sectioned surface, and aggrecan diffusional loss from the section are some of those issues. In our experiments, aggrecan loss was minimized by mild fixation which, however, did not prevent thickness changes of the cut sections. We performed high-resolution (1um) elasticity mapping of the cartilage matrix of newborn mouse across tissue depth. The indentations were performed on thin (∼12um) cartilage sections cut parallel to the bone longitudinal axis. We optimized the size of the spherical indentation probe (5um) and applied relatively deep indentations. We developed a novel data analysis method that determines a virtual contact point, and a fitting protocol that ensures that we are probing the bulk matrix properties and are minimizing the effects of surface roughness, tissue inhomogeneity and corrected for the finite sample thickness. We demonstrated the robustness and effectiveness of our method and we compared its performance to the standard methods used in the field. Thickness changes upon sectioning, and the high cell density, characteristic of mouse cartilage, that leaves narrow matrix septa between cells are two factors that may cause underestimation of the measured elastic moduli. Fixation is possibly causing over-estimation. We discuss the implications of these uncertainties and future directions to address them.

OI is a clinically and genetically heterogeneous disorder characterized by bone fragility. More than 90% of patients are heterozygous for mutations in type I collagen genes, COL1A1 and COL1A2, and a common mutation is substitution for an obligatory glycine in the triple helical Gly-X-Y repeats. Few non-glycine substitutions in the triple helical domain have been reported; most result in Y-position substitutions of arginine by cysteine. Here, we investigated leucine and cysteine substitutions for one Y-position arginine, p.Arg958 (Arg780 in the triple helical domain) of proα1(I) chains that cause mild OI. We compared their effects with two substitutions for glycine located in close proximity. Like substitutions for glycine, those for arginine reduced the denaturation temperature of the whole molecule and caused asymmetric posttranslational overmodification of the chains. Circular dichroism and increased susceptibility to cleavage by MMP1, MMP2 and catalytic domain of MMP1 revealed significant destabilization of the triple helix near the collagenase cleavage site. On a cellular level, we observed slower triple helix folding and intracellular collagen retention, which disturbed the Endoplasmic Reticulum function and affected matrix deposition. Molecular dynamic modeling suggested that Arg780 substitutions disrupt the triple helix structure and folding by eliminating hydrogen bonds of arginine side chains, in addition to preventing HSP47 binding. The pathogenic effects of these non-glycine substitutions in bone are probably caused mostly by procollagen misfolding and its downstream effects.

Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like lesions (FDLL) in mice, as well as isolated myxomas in humans may be caused by inactivation of PRKAR1A, the gene coding for the type 1a regulatory subunit (R1 a) of cAMP dependent protein kinase (PKA). OMXs and FDLL in mice lacking Prkar1 alpha grow from abnormal proliferation of adult bone stromal cells (aBSCs). Prkarla and Prkaca (coding for C alpha) haploinsufficiency leads to COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of aBSC5. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. We hypothesized that COX-2 inhibition may have an effect in FD and FDLL. In vitro treatment of a human cell line prepared from a FD patient with Celecoxib resulted in decreased PGE2 and cell proliferation. Treatment of mice haploinsufficient for R1 alpha and C alpha with 1500 mg/ kg Celecoxib led to decreased PGE2 and proliferation and increased apoptosis, with a corresponding gene expression profile, resulting in dramatic reduction of tumor growth. Furthermore, the treatment improved the organization of cortical bone that was adjacent to the tumor. We conclude that, in vitro and in vivo, Celecoxib had an inhibitory effect on FD cell proliferation and in mouse FDLL structure, respectively. We speculate that COX-2 inhibitors offer an attractive alternative to current treatments for benign tumors such as OMX and FD that, apart from tumor suppression, may mechanically stabilize affected bones. Published by Elsevier Ireland Ltd.

Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen triple helix and cause severe bone fragility and malformations (osteogenesis imperfecta [OI]). However, these mutations do not elicit the expected endoplasmic reticulum (ER) stress response, in contrast to other protein-folding diseases. Thus, it has remained unclear whether cell stress and osteoblast malfunction contribute to the bone pathology caused by Gly substitutions. Here we used a mouse with a Gly610 to cysteine (Cys) substitution in the procollagen α2(I) chain to show that misfolded procollagen accumulation in the ER leads to an unusual form of cell stress, which is neither a conventional unfolded protein response (UPR) nor ER overload. Despite pronounced ER dilation, there is no upregulation of binding immunoglobulin protein (BIP) expected in the UPR and no activation of NF-κB signaling expected in the ER overload. Altered expression of ER chaperones αB crystalline and HSP47, phosphorylation of EIF2α, activation of autophagy, upregulation of general stress response protein CHOP, and osteoblast malfunction reveal some other adaptive response to the ER disruption. We show how this response alters differentiation and function of osteoblasts in culture and in vivo. We demonstrate that bone matrix deposition by cultured osteoblasts is rescued by activation of misfolded procollagen autophagy, suggesting a new therapeutic strategy for OI. © 2016 American Society for Bone and Mineral Research.

Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated with bone lesions such as osteochondromyxomas (OMX). The most frequent cause for CNC is PRKAR1A deficiency; PRKAR1A codes for type-I regulatory subunit of protein kinase A (PKA). Prkar1a(+/-) mice developed OMX, fibrous dysplasia-like lesions (FDL) and other tumors. Tumor tissues in these animals had increased PKA activity due to an unregulated PKA catalytic subunit and increased PKA type II (PKA-II) activity mediated by the PRKAR2A and PRKAR2B subunits. To better understand the effect of altered PKA activity on bone, we studied Prkar2a and Prkar2b knock out (KO) and heterozygous mice; none of these mice developed bone lesions. When Prkar2a(+/-) and Prkar2b(+/-) mice were used to generate Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) animals, bone lesions formed that looked like those of the Prkar1a(+/-) mice. However, better overall bone organization and mineralization and fewer FDL lesions were found in both double heterozygote groups, indicating a partial restoration of the immature bone structure observed in Prkar1a(+/-) mice. Further investigation indicated increased osteogenesis and higher new bone formation rates in both Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) mice with some minor differences between them. The observations were confirmed with a variety of markers and studies. PKA activity measurements showed the expected PKA-II decrease in both double heterozygote groups. Thus, haploinsufficiency for either of PKA-II regulatory subunits improved bone phenotype of mice haploinsufficient for Prkar1a, in support of the hypothesis that the PRKAR2A and PRKAR2B regulatory subunits were in part responsible for the bone phenotype of Prkar1a(+/-) mice.

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.

Hyaline cartilage cushions bone at articular joints and is responsible for elongation and mechanical integrity of growing bones. The major components of cartilage matrix are the collagen mesh and the highly charged proteoglycan aggrecan entangled in and swelling the mesh. In growing cartilage, the composition and organization of these components varies across both growth plate and articular cartilage and in the vicinity of chondrocytes. Mice are extensively used as model system for a wide range of pathologies including arthritis, but there are limited studies mapping matrix nanomechanics across different regions. Nanomechanical studies, typically performed by indenting tissue sections, are subject to errors due to matrix collapse, surface roughness and diffusional loss aggrecan loss from the section surface. Moreover, there are often issues of data interpretation when the probe size and indentation depth are comparable to the mesh and aggrecan sizes. We performed a high-resolution (∼1um) nanoindentation elasticity mapping of extracellular matrix material of mouse growing cartilage across different regions of both cartilage types in physiological solution. We used an AFM with microsphere probes whose radii and indentation depth exceeded mesh and molecular sizes. We mildly fixed the cartilage to prevent aggrecan loss. We developed a new method for obtaining the Hertzian contact points on rough surfaces. We applied large-strain indentations and force corrections to remove the error due to surface roughness and to select the linear regime of the force data. The latter greatly improved the consistency of our elasticity results. Tissue collapse was partially corrected using the cartilage height mapping. Matrix regions were extracted by correlation with optical images. The matrix elasticity across the growing cartilage showed interesting difference between the two cartilage types and correlations with previous studies of matrix composition at the same locations.