Figures and data
Osx obviously expressed in osteocytes while Col1α1-creER labeled osteocytes.
(A) Immunofluorescence staining demonstrated the co-expression of Col1α1 and Osx in osteocytes of mouse cortical bone. Col1α1 = Collagen I α 1. Scale bar, 200 μM.
(B) The breeding scheme and genotyping for Osx conditional knockout mice. Het, heterozygote.
(C and D) Western blots showed the expression of Osx was repressed by small interfering RNA (si-Osx). The data represented the mean ± SD. Protein levels were normalized to β-actin. *p < 0.05.
(E) qPCR showed the mRNA expression of Osx in osteocyte was significantly increased after being transfected with lenti-Osx. Expression levels were normalized using GAPDH and lenti-GFP as controls. *p < 0.05.
Osx deficiency impaired cortical bone structure and decreased osteocytic dendrites both in vivo and in vitro.
(A) Immunohistochemical staining showed a decreased expression of Osx in osteocytes of the cortical bone in Osx conditional knockout (OsxcKO) mice when compared to wild-type (WT) group. Scale bar, 50 μM.
(B) The reduced expression of Osx in OsxcKO mice was confirmed via quantitative analysis as depicted in (A). The results were presented as the mean ± standard deviation (SD). The data were derived from multiple independent biological samples. *p < 0.05.
(C) Representative micro-computed tomography (μCT) images highlighted the differences in tibial cortical bone structure between OsxcKO mice and WT mice.
(D and E) Scanning electron microscopy (SEM) images demonstrated a reduction in osteocytic dendrites in the femurs of OsxcKO mice compared to WT mice. The white box delineated the dendritic changes of an individual osteocyte, while the red box showed alterations in intercellular dendrites among osteocytes. The number of dendrites was quantified per osteocyte, with a total of 16 cells analyzed per group. Scale bar, 50 μM & 20μM. *p < 0.05.
(F and G) SEM images revealed a similar reduction in osteocytic dendrites in the cranium bone of OsxcKO mice compared to WT mice. The white and red boxes served the same purpose as described in (D&E), and the same quantification method was applied. Scale bar, 50 μM & 20μM.
(H and I) SEM images showed a decrease in intercellular dendritic connections of osteocytes in the si-Osx group, where Osx expression was suppressed by si-Osx, compared to the control group. The number of dendrites was calculated per cell, with 16 cells analyzed each group. Scale bar, 50 μM & 20μM. *p < 0.05.
(J and K) SEM images demonstrated an increase in intercellular dendritic connections of osteocytes in the Lenti-Osx group, where Osx expression was over-expressed using a lentiviral vector, compared to the control group. The quantification of dendrites was performed per cell, with 16 cells analyzed per group. *p < 0.05. Scale bar, 50 μM & 20μM.
Osx deficiency compromised the transcellular fluid flow among osteocytes.
(A and B) Western blots confirmed a significant reduction in Osx expression in OsxcKO mice. n = 3. *p < 0.05.
(C and D) Primary pre-osteocytes, differentiated from osteoblasts using osteogenic induction medium, were assessed for intercellular signaling communication using a Lucifer Yellow Dye Transfer Assay, which involved loading the cells with 1 mg/ml Lucifer Yellow dye for 10 minutes. A significant reduction in transcellular fluid flow to approximately 40% of control levels was observed in the absence of Osx. n = 3. Scale bar, 150 μM. *p < 0.05.
(E) The effect shown in (C&D) was further visualized under a 60x fluorescence microscope, providing a clearer and more detailed view of the diminished gap junction channels. White arrows highlighted the reduced intercellular communication. Scale bar, 40 μM.
(F and G) Mlo-y4 osteocytes were evaluated in a manner similar to (C&D), comparing the si-Osx group with the control group. n = 3. *p < 0.05. Scale bar, 150 μM.
(H) The diminished gap junction in the si-Osx group was further visualized under a 60x fluorescence microscope, offering a detailed depiction of the reduced intercellular communication. White arrows indicated the areas where gap junction channels are notably decreased. Scale bar, 40 μM.
Osx deficiency reduced integrinαvβ1 expression of osteocytes.
(A) A heatmap provided a comparative analysis of mRNA expression levels of the integrin family in osteocytes between the lenti-Osx group and the control group.
(B and C) Western blots were used to assess the expression of integrinαvβ1 in Mlo-y4 osteocytes with si-Osx. The statistical analysis was normalized to the control group. n = 3. *p < 0.05.
(D and E) Western blots were conducted to evaluate the expression of integrinαvβ1 in Mlo-y4 osteocytes with lenti-Osx. The statistical analysis was normalized to the control group. n = 3. *p < 0.05.
(F) Immunofluorescence staining illustrated a reduction in integrinαvβ1 expression at both the osteocyte body and its intercellular dendrites with Osx deficiency. The cytoskeleton was stained green by F-actin, integrinαvβ1 was stained red, and nuclei were stained blue. n = 3. Scale bar, 40 μM.
(G) Immunofluorescence staining conversely showed an increase in integrinαvβ1 expression at the osteocyte body and its intercellular dendrites with the introduction of Lenti-Osx. The cytoskeleton, integrinαvβ1, and nuclei were stained as described in (F). n = 3. Scale bar, 40 μM.
Cx43 was the transcriptional target of Osx in osteocytes.
(A) The plot displayed the differences in expression peaks between the lenti-Osx group and the control group.
(B) Gene Ontology (GO) analysis was conducted on the differential expression peaks identified after performing CHIP-seq experiments with Osx. The analysis suggested that Osx played a regulatory role in multiple signaling pathways within osteocytes.
(C) GO analysis of mRNA-seq data indicated that the functions of Osx were predominantly associated with dendrite-related processes in osteocytes.
(D) A heatmap illustrated the changes in expression levels of the gap junction protein family (GJA) in osteocytes with Lenti-Osx as determined by mRNA-seq analysis.
(E) CHIP-seq analysis identified two distinct peaks in the Cx43 promoter region binding with Osx, highlighting the direct transcriptional regulation of Cx43 by Osx.
Osx promoted Cx43 expresion of osteocytes both in vitro and in vivo.
(A and B) In vivo immunohistochemical staining revealed a diminished Cx43 expression in the cortical bone of OsxcKO mice as opposed to the WT mice. Statistical analysis was performed using Image J software with normalization to the control group. Scale bar, 50 μM. *, p < 0.05.
(C and D) Western blots showed a decrease in Cx43 expression of OsxcKO pre- osteocytes. n = 3. Statistical analysis was based on the control group after normalization. *p < 0.05.
(E) qPCR showed the mRNA levels of Cx43 in OsxcKO pre-osteocytes. n = 3. *p < 0.05.
(F) Immunofluorescence staining depicted a diminished expression of Cx43 in OsxcKO pre-osteocytes relative to the WT group. The cytoskeleton (F-actin) was visualized in green, Cx43 in red, and nuclei in blue. n = 3. Scale bar, 40 μM.
(G, H and I) Western blots showed Cx43 expression level in Mlo-y4 osteocytes with lenti-Osx. Statistical comparisons were made relative to the control group after normalization. n=3. *p < 0.05.
(J) qPCR corroborated the increase of Cx43 mRNA in Mlo-y4 osteocytes with lenti-Osx. n=3. *p < 0.05.
(K) Immunofluorescence staining demonstrated an elevated Cx43 expression at the gap junction of osteocytes in the Lenti-Osx group
(L) compared to the WT group. The images showed GFP in green, Cx43 in red, and nuclei in blue. n=3. Scale bar, 40 μM.
Restoration of Cx43 rescued intercellular communication among Osx- deficient osteocytes both in vivo and in vitro.
(A) Western blots was employed to assess the impact of all-trans-retinoic acid (ATRA), a Cx43 signaling agonist, on Cx43 expression in Mlo-y4 osteocytes. n = 3.
(B and C) The Lucifer Yellow Dye Transfer Assay was utilized to evaluate the gap junction intercellular communication among osteocytes. ATRA intervention significantly restored the impaired communication observed in Osx-deficient osteocytes. n = 3. Scale bar, 150 μM.
(D and E) Scanning electron microscopy (SEM) was conducted to visualize the osteocytic dendrites in the cranial bone. The results demonstrated that ATRA intervention effectively reversed the reduction in dendritic density of osteocytes observed in OsxcKO mice. Dendrite counts were performed per osteocyte, with 16 osteocytes analyzed per group. Scale bar, 50 μM & 20 μM. *p < 0.05.
(F) Western blots revealed a decrease in integrinαvβ1 expression in Mlo-y4 osteocytes with si-Osx. By contrast, ATRA treatment rescued the reduced integrinαvβ1 expression.
(G) Immunofluorescent staining corroborated the Western blots findings, showing that ATRA intervention restored integrinαvβ1 expression in osteocytes with si-Osx. The cytoskeleton (F-actin) was visualized in green, integrinαvβ1 in red, and nuclei in blue. Scale bar, 40 μM.
Schematic model illustrated the Osx-Cx43 axis of osteocytes and its role in intercellular signaling communication.
The dendrites of osteocytes were essential for maintaining gap junction intercellular communication, given that osteocytes were non-migratory, as embedded within the bone mineralized matrix.
(A) In physiological state, Osx directly promoted the transcription of Cx43, the key component of gap junction channel, which was a critical regulatory mechanism that preserved the integrity and function of osteocytic dendrites, thus governing the transmission of signals among osteocytes within the mature bone tissue.
(B) In pathological state, Osx deficiency inhibited the Cx43 expression, then restrained the dendritic network among osteocytes, thereby impairing osteocyte in response to environmental stimuli and signal transduction, such as mechanical forces or bone fracture.
The expression of Cx43 was reduced in Osx-deficient osteocytes both in vitro and in vivo.
(A) Immunofluorescence staining substantiated the decreased expression of Cx43 in the OsxcKO mice. Scale bar, 500 μM.
(B and C) Western blots revealed a reduction in Cx43 expression in Mlo-y4 osteocytes transfected with si-Osx. n = 3, with statistical analysis normalized to the control group. * p < 0.05.
(D) qPCR showed the mRNA levels of Cx43 decreased in si-Osx Mlo-y4 osteocytes. n = 3. *p < 0.05.
(E) Immunofluorescence staining showed a reduced expression of Cx43 in Mlo-y4 osteocytes with si-Osx as compared to the control group. The cytoskeleton was marked in green, Cx43 in red, and nuclei in blue. n = 3. Scale bar, 40 μM.
