Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Nov 23;24(23):16648.
doi: 10.3390/ijms242316648.

Genetic Deficiency of the Long Pentraxin 3 Affects Osteogenesis and Osteoclastogenesis in Homeostatic and Inflammatory Conditions

Valentina Granata  1 , Dario Strina  1   2 , Maria Lucia Schiavone  1 , Barbara Bottazzi  1 , Alberto Mantovani  1   3   4 , Antonio Inforzato  1   3 , Cristina Sobacchi  1   2
Affiliations

Genetic Deficiency of the Long Pentraxin 3 Affects Osteogenesis and Osteoclastogenesis in Homeostatic and Inflammatory Conditions

Valentina Granata et al. Int J Mol Sci. .

Abstract

The long pentraxin 3 (PTX3) is a soluble glycoprotein made by immune and nonimmune cells endowed with pleiotropic functions in innate immunity, inflammation, and tissue remodeling. PTX3 has recently emerged as a mediator of bone turnover in both physiological and pathological conditions, with direct and indirect effects on osteoblasts and osteoclasts. This notwithstanding, its role in bone biology, with major regard to the osteogenic potential of osteoblasts and their interplay with osteoclasts, is at present unclear. Here, we investigated the contribution of this pentraxin to bone deposition in the osteogenic lineage by assessing collagen production, mineralization capacity, osteoblast maturation, extracellular matrix gene expression, and inflammatory mediators' production in primary osteoblasts from the calvaria of wild-type (WT) and Ptx3-deficient (Ptx3-/-) mice. Also, we evaluated the effect of PTX3 on osteoclastogenesis in cocultures of primary osteoblasts and bone marrow-derived osteoclasts. Our investigations were carried out both in physiological and inflammatory conditions to recapitulate in vitro aspects of inflammatory diseases of the bone. We found that primary osteoblasts from WT animals constitutively expressed low levels of the protein in osteogenic noninflammatory conditions, and genetic ablation of PTX3 in these cells had no major impact on collagen and hydroxyapatite deposition. However, Ptx3-/- osteoblasts had an increased RANKL/OPG ratio and CD44 expression, which resulted in in enhanced osteoclastogenesis when cocultured with bone marrow monocytes. Inflammation (modelled through administration of tumor necrosis factor-α, TNF-α) boosted the expression and accumulation of PTX3 and inflammatory mediators in WT osteoblasts. In these conditions, Ptx3 genetic depletion was associated with reduced collagen deposition and immune modulators' production. Our study shed light on the role of PTX3 in osteoblast and osteoclast biology and identified a major effect of inflammation on the bone-related properties of this pentraxin, which might be relevant for therapeutic and/or diagnostic purposes in musculoskeletal pathology.

Keywords: PTX3; bone; hyaluronan-rich matrix; osteoblast; osteoclast; tissue remodeling.

PubMed Disclaimer

Conflict of interest statement

All the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effect of Ptx3 genetic deficiency on the osteogenic properties of OBs. (A) Analysis of Ptx3 expression in primary OBs from calvariae of WT mice in basal condition and at different timepoints during osteogenic induction. Ptx3 mRNA levels were determined by qPCR. Results were normalized based on Gapdh expression and reported as arbitrary units (AU); * p < 0.05. (B) PTX3 protein was quantified in the supernatant of WT OB cultures by ELISA (3 independent experiments performed in duplicate, n = 6, mean ± SEM). One-way ANOVA with multiple comparison test. (C) Assessment of in vitro collagen production by WT and Ptx3−/− OBs stimulated with ascorbic acid for 8 h. Cultures were stained with Sirius red; representative images are shown. Afterwards, staining was extracted, and absorbance measured at 540 nm. Mann–Whitney test. (D) Time-course analysis of mineralization in WT and Ptx3−/− primary OB cultures, as assessed by Alizarin red staining (ARS) and quantification at 405 nm and expression of osteogenic marker genes. One-way ANOVA with multiple comparison test; * p < 0.05. All the data presented in this figure are derived from 3 independent experiments performed in duplicate, n = 6, and are represented as mean ± SEM.
Figure 2
Figure 2
Effect of Ptx3 genetic deficiency on OB-induced osteoclastogenesis. (A) RANKL/OPG ratio in the supernatant of WT and Ptx3−/− OB cultures in basal conditions and at different timepoints of osteogenic induction. RANKL and OPG were quantified using dedicated ELISA kits (3 independent experiments performed in duplicate, n = 6, mean ± SEM). Two-way ANOVA with multiple comparison test; * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Schematic representation of the protocol for coculture experiments. (C) Representative images of TRAP-stained OCs formed after coculture of WT or Ptx3−/− OBs with WT bone marrow cells for 7 days. Images were taken with an EVOS microscope at 20× magnification; scale bar: 200 μm. TRAP+ multinucleated (nuclei number ≥ 3) OCs were counted by a blind operator and expressed as number of mature OCs per well. Mann–Whitney test; ** p < 0.01. (D) RANKL, OPG, and RANKL/OPG ratio in the supernatant of the cocultures described in C. The conditioned medium was collected from each well after 7 days of coculture (2 independent experiments performed in triplicate, n = 6, mean ± SEM). Mann–Whitney test; * p < 0.05. (E) Analysis of M-csf expression in the coculture setting described in (C) after 7 days of coculture; mRNA levels were determined by qPCR. Results were normalized based on Gapdh expression and expressed as arbitrary units (AU). Mann–Whitney test.
Figure 3
Figure 3
(A) FACS analysis of the indicated surface markers on WT and Ptx3−/− KO OBs in basal condition (not treated with oim). The percentage of CD45- cells expressing each marker as well as the mean fluorescence intensity for each marker in both genotypes is indicated as mean ± SEM. Mann–Whitney test; * p < 0.05. (B) Gene expression analysis of the hyaluronic acid synthases Has1, Has2, and Has3 and Tnfaip6 gene in WT and Ptx3−/− OBs at different timepoints during osteogenic induction; mRNA levels were determined by qPCR and normalized on Gapdh expression, and they are indicated as arbitrary units (AU) (3 independent experiments performed in duplicate, n = 6, mean ± SEM). One-way ANOVA.
Figure 4
Figure 4
Expression of immune mediators in WT and Ptx3−/− KO OBs at different timepoints during osteogenic induction. (A) Gene expression analysis of selected factors (as indicated in the graphs) known to attract and regulate both immune cells and skeletal cells and their progenitors; mRNA levels were determined by qPCR and normalized on Gapdh expression, and they are indicated as arbitrary units (AU) (3 independent experiments performed in duplicate, n = 6, mean ± SEM). One-way ANOVA. (B) Protein expression of selected factors (as indicated in the graphs) in cell lysates of WT and Ptx3−/− OBs. Protein levels were measured using a highly sensitive immunoassay and normalized on the total amount of proteins in the cell lysates (n ≥ 3, mean ± SEM). Two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
Effect of Ptx3 genetic deficiency on OB functions in the presence of inflammation. (A) Assessment of in vitro collagen production by WT and Ptx3−/− OBs treated with TNF-α for 24 h prior to stimulation with ascorbic acid. Cultures were stained with Sirius red; representative images are shown here. Afterwards, staining was extracted, and absorbance measured at 540 nm. Mann–Whitney test; * p < 0.05. (B) Schematic representation of the experimental protocol for osteogenic induction in the presence of TNF-α. CM = conditioned medium. (C) Analysis of Ptx3 expression in WT OBs in basal condition and at different timepoints during osteogenic induction in the presence of TNF-α. One-way ANOVA with multiple comparison test; ** p < 0.01. (D) WT OBs were lysed at 0, 7, and 14 days of osteogenic induction in the absence and presence of TNF-α. Lysate samples containing 10 μg total proteins were analyzed by Western blotting using an anti-mouse PTX3 polyclonal antibody. Purified recombinant mouse PTX3 (rmPTX3) was used as a reference (1, 2, and 4 ng/lane). A representative gel is shown with indication of the apparent molecular weight of the protein standards assigned following the manufacturer’s instructions for NuPAGE Bis-Tris 4–12% with MES buffer (Life Technologies). In these gels, PTX3 is detected as two immune reactive bands with apparent molecular weights of 42 (1-mer) and 84 (2-mer) kDa [4]. (E) PTX3 protein in the supernatant of WT OB cultures treated with TNF-α during osteogenic induction was quantified by ELISA. One-way ANOVA with multiple comparison test. (F) Time-course analysis of mineralization and expression of osteogenic marker genes by WT and Ptx3−/− KO primary OB cultures in the presence of TNF-α. (G) Time-course analysis of RANKL/OPG ratio in the supernatant of WT and Ptx3−/− KO primary OB cultures in the presence of TNF-α during osteogenic induction. All the data presented in this figure (except Western blot) are derived from 3 independent experiments performed in duplicate, n = 6, and are represented as mean ± SEM.
Figure 6
Figure 6
(A) Expression of Ha synthase isoforms and Tnfaip6 in WT and Ptx3−/− KO OBs at different timepoints during osteogenic induction in inflammatory condition. (B) Gene expression analysis of selected immune regulatory factors (as indicated in the graphs). In (A,B), mRNA levels were determined by qPCR and normalized on Gapdh expression, and they are indicated as arbitrary units (AU) (3 independent experiments performed in duplicate, n = 6, mean ± SEM). One-way ANOVA. (C) Protein expression of selected factors (as indicated in the graphs) in cell lysates of WT and Ptx3−/− OBs. Protein levels were measured by immunoassay and normalized on the total amount of proteins in the cell lysates (n ≥ 3, mean ± SEM). Statistical analysis using two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 7
Figure 7
Schematic representation of the mechanism of Ptx3 regulation of OB and OC formation and function in homeostatic and inflammatory conditions. Left: In conditions of bone homeostasis, PTX3 is synthesized by pre- and mature OBs (1); the released protein inhibits RANKL production (2) and stimulates OPG release (3). This results in reduced RANKL/OPG ratio, which helps restraining osteoclastogenesis (4). A similar effect might be contributed to by the PTX3-dependent control of CD44 expression on OBs (5). Right: Inflammatory conditions (i.e., TNF-α stimulation) increase PTX3 expression in OBs (6). The newly synthesized protein, possibly via the HA network and the engagement of CD44 (7), stimulates collagen production and deposition by OBs (8). At the same time, TNF-α has direct osteoclastogenic effects on OC precursors (9). Also, PTX3 is required for OBs to make and release inflammatory chemokines like CCL2 and CCL3 (10). Created with BioRender.com.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Garlanda C., Bottazzi B., Magrini E., Inforzato A., Mantovani A. PTX3, a humoral pattern recognition molecule, in innate immunity, tissue repair, and cancer. Physiol. Rev. 2018;98:623–639. doi: 10.1152/physrev.00016.2017. - DOI - PMC - PubMed
    1. Porte R., Silva-Gomes R., Theroude C., Parente R., Asgari F., Sironi M., Pasqualini F., Valentino S., Asselta R., Recordati C., et al. Regulation of inflammation and protection against invasive pneumococcal infection by the long pentraxin PTX3. eLife. 2023;12:e78601. doi: 10.7554/eLife.78601. - DOI - PMC - PubMed
    1. Parente R., Sobacchi C., Bottazzi B., Mantovani A., Grčevic D., Inforzato A. The Long Pentraxin PTX3 in Bone Homeostasis and Pathology. Front. Immunol. 2019;10:2628. doi: 10.3389/fimmu.2019.02628. - DOI - PMC - PubMed
    1. Massimino A.M., Colella F.E., Bottazzi B., Inforzato A. Structural insights into the biological functions of the long pentraxin PTX3. Front. Immunol. 2023;14:1274634. doi: 10.3389/fimmu.2023.1274634. - DOI - PMC - PubMed
    1. Noone D.P., Dijkstra D.J., van der Klugt T.T., van Veelen P.A., de Ru A.H., Hensbergen P.J., Trouw L.A., Sharp T.H. PTX3 structure determination using a hybrid cryoelectron microscopy and AlphaFold approach offers insights into ligand binding and complement activation. Proc. Natl. Acad. Sci. USA. 2022;119:e2208144119. doi: 10.1073/pnas.2208144119. - DOI - PMC - PubMed

LinkOut - more resources