Cytopathologic Diagnosis of Serous Fluids
cflogo
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Filter by Categories
Abstracts
Book Review
Case Report
Case Series
CMAS‡ - Pancreas - EUS-FNA Cytopathology (PSC guidelines) S1:1 of 5
CMAS‡ - Pancreas - EUS-FNA Cytopathology (PSC guidelines) S1:3 of 5
CMAS‡ - Pancreas - EUS-FNA Cytopathology (PSC guidelines) S1:4 of 5
CMAS‡ - Pancreas -Sampling Techniques for Cytopathology (PSC guidelines) S1:2 of 5
CMAS‡ - Pancreas- EUS-FNA Cytopathology (PSC guidelines) S1:5 of 5
Commentary
Correction
CytoJournal Monograph Related Review Series
CytoJournal Monograph Related Review Series (CMAS), Editorial
CytoJournal Monograph Related Review Series: Editorial
Cytojournal Quiz Case
Editorial
Erratum
Letter to Editor
Letter to the Editor
Letters to Editor
Methodology
Methodology Article
Methodology Articles
Original Article
Pap Smear Collection and Preparation: Key Points
Quiz Case
Research
Research Article
Review
Review Article
Systematic Review and Meta Analysis
View Point
View/Download PDF

Translate this page into:

Research Article
2026
:23;
11
doi:
10.25259/Cytojournal_36_2025

Integrin β1 in osteoblast differentiation and migration during acute bone loss after fracture: Regulation of extracellular signal-regulated kinase 1/2 signaling pathway

, ,
1Trauma Department II, Yantai Mountain Hospital East, Yantai, China
2Medical Laboratory, Qingdao Municipal Hospital, Qingdao, China
3Trauma Department of Orthopedics, Yantaishan Hospital, Yantai, Shandong, China.
Author image

*Corresponding author: Jianhang Wang, Trauma Department of Orthopedics, Yantaishan Hospital, Yantai, Shandong, China. wangjianhang_yts@163.com

Received: , Accepted: ,
© 2026 The Author(s). Published by Scientific Scholar
Licence
This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Luan J, Li J, Wang J. Integrin β1 in osteoblast differentiation and migration during acute bone loss after fracture: Regulation of extracellular signal-regulated kinase 1/2 signaling pathway. CytoJournal. 2026;23:11. doi: 10.25259/Cytojournal_36_2025

Abstract

Objective:

Integrin β1 (ITGB1) reportedly participates in osteoblast differentiation, mineralization, and migration. Nevertheless, its role and underlying mechanism in osteoblast differentiation and migration during acute bone loss after fracture are not yet clear. This research was designed to measure the role of ITGB1 in osteoblast differentiation and migration and the underlying mechanism.

Material and Methods:

ITGB1 expression was assessed in MC3T3-E1 cells at different incubation times (0, 4, 7, 14, 21, and 28 days) through quantitative real-time polymerase chain reaction. Alkaline phosphatase (ALP) activity determination, ALP staining, and Alizarin red staining were performed to evaluate the differentiation degree of osteoblasts. Western blot was performed to detect the expression of markers related to osteoblast differentiation. Osteoblast migration ability was determined by wound healing and transwell assays. The molecular mechanism by which ITGB1 modulated the differentiation and migration of osteoblasts was examined by Western blot.

Results:

ITGB1 expression increased significantly after 14, 21, and 28 days of osteoblast differentiation. ITGB1 increases enhanced osteoblast differentiation and migration; conversely, reduced ITGB1 inhibits osteoblast differentiation and migration. Mechanically, ITGB1 facilitated the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, and the suppression of the ERK ERK1/2 pathway attenuated the effects of ITGB1 on osteoblast differentiation and migration.

Conclusion:

ITGB1 plays an important role in osteoblast differentiation and migration by activating the ERK1/2 signaling pathway, which may provide novel insights into bone injury treatment.

Keywords

Differentiation
Extracellular signal-regulated kinase 1/2
Integrin β1
Migration
Osteoblast

INTRODUCTION

Acute bone loss typically occurs within 1–2 years following fractures, with a particularly pronounced decline during the first 6–8 weeks after acute immobilization. The patient’s bone mass experiences a significant reduction, reaching its lowest level.[1] The phenomenon of acute bone loss post-fracture is prevalent, especially among individuals with osteoporosis.[2] This clinical challenge is closely linked to osteoporosis in terms of pathogenesis and therapeutic approaches. Osteoporosis is an age-related metabolic disorder characterized by decreased bone mass, deteriorated bone microarchitecture, and heightened susceptibility to fractures.[3] Bone-fracture healing is a complex physiological process involving many kinds of cells, requiring reconstruction under the coordination of osteoblasts, osteoclasts, various regulatory factors, and extracellular matrix to form new bone.[4,5] Osteoblasts, derived from pluripotent bone marrow stromal cells, are the main functional cells of osteogenesis and bone formation, responsible for the synthesis and secretion of bone stroma.[6,7] Osteoblasts, as direct osteogenic cells in the body, migrate to specific sites and differentiate into bone cells, subsequently secreting bone matrix to promote matrix mineralization and form bone tissue.[8,9] Therefore, the differentiation and migration of osteoblasts have a very important influence on the bone-healing process. Thus, understanding the functions and molecular regulatory mechanisms of osteoblasts can enable the effective treatment of this disease.

Numerous studies have shown that post-fracture reconstruction is regulated by intracellular signaling pathways, such as extracellular signal-regulated kinase (ERK), Wnt, and bone morphogenetic protein signaling pathways.[10-12] Extracellular signal-regulated kinase 1/2 (ERK1/2), a serine and threonine kinase, has different functions at different locations in the cell. ERK1/2 in local adhesion is well known to participate in cell-migration modulation.[13-15] Kwak et al. further demonstrated that ERK1/2 activation is strongly associated with osteogenesis.[16] Mehrotra et al. found that PD98095, an inhibitor of the ERK pathway, inhibits the migration of osteoblasts induced by platelet-derived growth factor.[17] Lee et al. discovered that ERK participates in osteoblast differentiation induced by eucalyptol in vivo and in vitro.[18] Park et al. reported that suppressing ERK phosphorylation blocks the osteoblast differentiation induced by the overexpression of runt-related transcription factor 2.[19] These findings suggest that ERK1/2 plays a strong part in modulating the differentiation and migration of osteoblasts.

Integrin is a kind of surface-adhesion cell molecule. It belongs to the membrane receptor family, whose main function is to mediate cell-cell and cell-extracellular matrix adhesion and signal transduction, thereby affecting cell adhesion, growth, differentiation, proliferation, and migration.[20,21] According to the difference of b subunits, integrins are divided into β1, β2, and β3 subgroups.[22] Integrin β1 (ITGB1), the largest of the integrin family, is an essential molecule for intercellular adhesion and is closely associated with the biological malignant behavior of many cells. ITGB1 is generally believed to positively regulate the cell cycle and promote cell proliferation.[23] ITGB1 deletion reportedly reduces the proliferation activity of mammary epithelial cells and pancreatic b cells.[24,25] ITGB1 on the surface of osteoblasts binds to fibronectin or type I collagenase in the extracellular matrix, mediating bidirectional signaling between the extracellular matrix and cells.[26] The proliferation of rat osteoblasts is significantly enhanced when ITGB1 expression is up-regulated.[27] However, the interaction between ITGB1 and ERK1/2 pathway in the regulation of osteoblast differentiation and migration remains poorly understood.

In the present study, we hypothesized that ITGB1 may interact with the ERK1/2 pathway to regulate the differentiation and migration of osteoblasts. The effects of ITGB1 and its interaction with ERK1/2 pathway on the differentiation and migration of osteoblasts were explored. Our findings may offer valuable insights into the development of new treatments for bone healing.

MATERIAL AND METHODS

Cell culture and differentiation induction

Mouse embryonic osteoblast cells MC3T3-E1 (cat. no. CL-0710) were purchased from Pricella (Wuhan, China). The cells tested negative for the presence of mycoplasma and were authenticated by short tandem repeat profiling. They were cultured in a-modified minimum essential medium (a-MEM; cat. no. PM150421; Pricella) containing 10% fetal bovine serum (FBS; cat. no. C0226; Beyotime, Shanghai, China) and 1% penicillin/streptomycin (cat. no. C0222; Beyotime) in a humid environment of 37°C and 5% CO2. To induce MC3T3-E1 cell differentiation, 1 × 105 cells were inoculated on a six-well plate and cultured in a-MEM (3 mL per plate) containing 10 mmol/L b-glycerophosphate (cat. no. ST637–2 g; Beyotime) and 50 μmol/L ascorbic acid (cat. no. ST1434–25 g; Beyotime). The medium was changed every 3 days.

Cell transfection

After trypsinization, MC3T3-E1 cells were implanted in 24-well plates. Transfection was conducted after cell fusion to a density of about 60–70%. pcDNA3.1-ITGB1 and small interfering RNA targeting ITGB1 (si-ITGB1) synthesized by GenePharma (Shanghai, China) were applied for ITGB1 overexpression and knockdown. Then, 5 × 104 MC3T3-E1 cells were implanted on 24-well plates (1,000 μL of a-MEM per plate) and transfected with pcDNA3.1-ITGB1, si-ITGB1, and their corresponding controls with lipofectamine 2,000 (Invitrogen, Carlsbad, CA, USA). Cells without any transfection served as the blank group. After 48 h, transfection efficiency was measured by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The primer sequences of si-ITGB1 and si-NC were as follows: si-ITGB1, forward, 5’-CGGAAAAGAUGAAUUUACATT-3’, reverse, 5’-UGUAAAUUCAUCUUUUCCGTT-3’; si-NC, forward, 5’-UUCUCCGAACGUGUCACGUTT’, reverse, 5’-ACGUGACACGUUCGGAGAATT-3’.

qRT-PCR

The total RNA of MC3T3-E1 cells at days 0, 4, 7, 14, 21, and 28 of induction was extracted using an RNA/DNA Co-isolation Kit (cat. no. R0017S; Beyotime), and RNA was reverse transcribed into cDNA with a reverse-transcription kit (cat. no. 330401; Qiagen, Dusseldorf, GER). PCR was performed according to the instructions of a QuantiTect SYBR Green PCR Kit (cat. no. 204145; Qiagen). ITGB1 was amplified by PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference, and three replicates were set for each sample. Relative expression was calculated by 2-∆∆Ct. The primer sequences used in qRT-PCR were as follows: ITGB1, forward, 5’-AAATGTAACCAACCGTAGC-3’, reverse, 5’-AGACAGGTCCATAAGGTAGTA-3’; GAPDH, forward, 5’-CAGGAGGCATTGCTGATGAT-3’, reverse, 5’-GAAGGCTGGGGCTCATTT-3’.

Western blot assay

Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysate (cat. no. P0013B; Beyotime), and protein concentration was determined by bicinchoninic acid method (cat. no. P0009; Beyotime). After adding ×5 loading buffer according to volume ratio, denaturation at 99°C for 10 min was performed. About 40 μg of protein/hole was added to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel for electrophoresis. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (cat. no. P0965–20 pcs; Beyotime) and then immersed in 5% skim milk for 1 h. The PVDF membranes were placed in an incubator box containing diluted primary antibodies, namely ITGB1 (cat. no. ab179471; Abcam, Cambridge, UK; 1:1000 dilution), osteocalcin (OCN; cat. no. ab309521; Abcam; 1:1000 dilution), osteopontin (OPN; cat. no. ab218237; Abcam; 1:1000 dilution), osteonectin (ON; cat. no. ab290636; Abcam; 1:1000 dilution), bone sialoprotein (BSP; cat. no. ab270605; Abcam; 1:1000 dilution), ERK1/2 (cat. no. ab184699; Abcam; 1:1000 dilution), p-ERK1/2 (cat. no. (ab201015; Abcam; 1:1000 dilution), and GAPDH (cat. no. ab8245; Abcam; 1:3000 dilution) at 4°C overnight, followed by secondary antibodies (cat. no. ab6721; Abcam; 1:1000 dilution) for 1 h. An enhanced chemiluminescence solution (cat. no. P0018S; Beyotime) was used to observe the Western blot. Image J 1.36b software (NIH, Bethesda, MD, USA) was used to analyze the gray values of the protein.

Alkaline phosphatase (ALP) activity assay

MC3T3-E1 cells (1 × 104) were inoculated on a 24-well plate (1,000 μL of a-MEM per plate). After 7 days of cell induction, 200 μL of lysis buffer was added, and the mixture was placed on ice for 5 min. After centrifuging the cells at 1,000 × g for 10 min and 4°C, ALP activity was detected using a LabAssay™ ALP kit (cat. no. ZWK-291–58601; ZZBIO Co., Ltd, Shanghai, China). Absorbance was measured at 405 nm with a microplate reader (DR-3518G, Hiwell Diatek, Wuxi, China).

ALP staining

MC3T3-E1 cells (1 × 104) per transfection group were washed twice with phosphate-buffered saline (PBS; cat. no. C0221A; Beyotime) on differentiation medium for 7 days and fixed with 10% paraformaldehyde for 10 min. After washing with PBS, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining solution (cat. no. C3206; Beyotime) was added, and the mixture was incubated for 30 min away from light, followed by neutral red dye solution for 3 min. After washing with distilled water, cell staining was observed under an inverted microscope (Axio Scope A1; Carl Zeiss, Oberkochen, Germany).

Alizarin red staining (ARS)

MC3T3-E1 cells (1 × 104) per transfection group were washed twice with PBS on differentiation medium for 7 days and fixed with 5% paraformaldehyde for 10 min. Subsequently, the fixed cells were stained with 1% ARS solution (cat. no. C0138; Beyotime) at 37°C for 30 min, and the formation of mineralized nodules was observed by optical microscopy. The rate of mineralized-nodule formation was assessed with Image J software (NIH, Bethesda, MD, USA).

Wound-healing assay

MC3T3-E1 cells (5 × 104) were inoculated on six-well culture plates (3 mL of a-MEM per plate). After the cells attached onto the wall with a single layer, a scratch of 1 mm wide was made at the bottom of the plate using a 200 μL gun tip to mark the observation site. After the shed cells were washed off with PBS, cell culturing was continued, and photographs were taken at 0 and 24 h, respectively. The relative width between two scratches was calculated.

Transwell assay

About 200 μL of MC3T3-E1 cell suspension was inoculated into the upper chamber of the Transwell chamber, and 500 μL of culture medium containing 10% FBS was added into the lower chamber. After 24 h of culture, the lower cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 10 min. The crystal violet-stained cells were calculated as the number of migrating cells.

Statistical analysis

All data were representative results, and similar data were duplicated at least 3 times. GraphPad Prism 8.0 software (Graphpad™, San Diego, USA) was used to analyze the experimental data, and a difference of P < 0.05 was considered statistically significant. All data are presented as the mean ± standard deviation. For comparison among multiple groups, one-way analysis of variance with Tukey’s multiple comparison test was used. For comparison between the two groups, Student’s t-test was used.

RESULTS

Over-expression of ITGB1 promoted osteoblast differentiation and migration

To probe into the functions of ITGB1 on osteoblast differentiation, ITGB1 expression was analyzed in the MC3T3-E1 cells at different incubation times (0, 4, 7, 14, 21, and 28 days) by qRT-PCR. As shown in Figure 1a, ITGB1 expression increased significantly after 14, 21, and 28 days of osteoblast differentiation. As shown in Figure 1b-d, ITGB1 levels notably increased in MC3T3-E1 cells after transfection with pcDNA3.1-ITGB1. This finding indicated the successful transfection of ITGB1 into cells. ALP activity assay results revealed that ITGB1 overexpression exhibited a more pronounced increase in ALP activity [Figure 1e]. Similarly, we found that the re-expression of ITGB1 increased the percentage of positive cells stained with ALP and ARS [Figure 1f-i]. Subsequently, the levels of osteogenic markers such as OCN, OPN, ON, and BSP were measured by Western blot. Our findings revealed that ITGB1 overexpression showed a more pronounced increase in these above levels [Figure 1j-n]. We further examined ITGB1 effects on osteoblast migration using wound healing and transwell assays. As shown in Figure 1o and p, the wound-healing rate of osteoblast was significantly elevated following transfection with pcDNA3.1-ITGB1. Similarly, transwell assay showed that ITGB1 re-expression signally enhanced osteoblast migration compared with the control group [Figure 1q and r]. These findings demonstrated that ITGB1 positively affected osteoblast differentiation and migration.

ITGB1 overexpression promoted osteoblast differentiation and migration. pcDNA3.1-ITGB1 was transfected into MC3T3-E1 cells. (a) ITGB1 expression in MC3T3-E1 cells on days 0, 4, 7, 14, 21, and 28. The transfection efficiency of ITGB1 was tested by (b and c) Western blot and (d) qRT-PCR. ALP activity was determined by (e) ALP activity assay and (f and g) ALP staining. Scale bar = 200 μm. (h and i) ARS staining was applied to evaluate the degree of osteoblast matrix mineralization. Scale bar = 200 μm. (j-n) The levels of osteogenic markers (OCN, OPN, ON, and BSP) were measured by Western blot. Osteoblast migration was measured by (o and p) wound healing (Scale bar = 100 μm) and (q and r) transwell assays (Scale bar = 50 μm). ✶P<0.05; ✶✶P<0.01; ✶✶✶P<0.001. n=3. ITGB1: Integrin β1, qRT-PCR: Quantitative real-time polymerase chain reaction, ALP: Alkaline phosphatase, ARS: Alizarin red staining, OCN: Osteocalcin, OPN: Osteopontin, ON: Osteonectin, BSP: Bone sialoprotein, mRNA: Messenger ribonucleic acid, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, pcDNA: Plasmid cloning deoxyribonucleic acid.
Figure 1:
ITGB1 overexpression promoted osteoblast differentiation and migration. pcDNA3.1-ITGB1 was transfected into MC3T3-E1 cells. (a) ITGB1 expression in MC3T3-E1 cells on days 0, 4, 7, 14, 21, and 28. The transfection efficiency of ITGB1 was tested by (b and c) Western blot and (d) qRT-PCR. ALP activity was determined by (e) ALP activity assay and (f and g) ALP staining. Scale bar = 200 μm. (h and i) ARS staining was applied to evaluate the degree of osteoblast matrix mineralization. Scale bar = 200 μm. (j-n) The levels of osteogenic markers (OCN, OPN, ON, and BSP) were measured by Western blot. Osteoblast migration was measured by (o and p) wound healing (Scale bar = 100 μm) and (q and r) transwell assays (Scale bar = 50 μm). ✶P<0.05; ✶✶P<0.01; ✶✶✶P<0.001. n=3. ITGB1: Integrin β1, qRT-PCR: Quantitative real-time polymerase chain reaction, ALP: Alkaline phosphatase, ARS: Alizarin red staining, OCN: Osteocalcin, OPN: Osteopontin, ON: Osteonectin, BSP: Bone sialoprotein, mRNA: Messenger ribonucleic acid, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, pcDNA: Plasmid cloning deoxyribonucleic acid.

Knockdown of ITGB1 inhibited osteoblast differentiation and migration

MC3T3-E1 cells were transfected with si-ITGB1 for loss-of-function experiments. As shown in Figure 2a-c, ITGB1 levels were notably reduced in MC3T3-E1 cells transfection with si-ITGB1. ALP activity assay revealed that ITGB1 deletion exhibited a more pronounced reduction in ALP activity [Figure 2d]. Similarly, ITGB1 lack decreased the percentage of positive cells stained with ALP and ARS [Figure 2e-h]. In addition, the levels of osteogenic markers were notably inhibited in MC3T3-E1 cells following treatment with si-ITGB1 [Figure 2i-m]. Furthermore, wound-healing results revealed that osteoblast wound healing significantly decreased after transfection with si-ITGB1 [Figure 2n and o]. Transwell assay also demonstrated that lack of ITGB1 signally suppressed osteoblast migration, when compared with the si-NC group [Figure 2p and q].

ITGB1 knockdown inhibited osteoblast differentiation and migration. MC3T3-E1 cells were transfected with si-ITGB1. The transfection efficiency of ITGB1 was tested by (a and b) Western blot and (c) qRT-PCR. ALP activity was determined by (d) ALP activity assay and (e and f) ALP staining. Scale bar = 200 μm. (g and h) ARS staining was applied to evaluate the degree of osteoblast matrix mineralization. Scale bar = 200 μm. (i-m) The levels of osteogenic markers (OCN, OPN, ON, and BSP) were measured by Western blot. Osteoblast migration was measured by (n and o) wound healing (Scale bar = 100 μm) and (p and q) transwell assays (Scale bar = 50 μm). ✶P<0.05; ✶✶P<0.01; ✶✶✶P<0.001. n=3. ITGB1: Integrin β1, qRT-PCR: Quantitative real-time polymerase chain reaction, ALP: Alkaline phosphatase, ARS: Alizarin red staining, OCN: Osteocalcin, OPN: Osteopontin, ON: Osteonectin, BSP: Bone sialoprotein, si-NC: Small interfering negative control, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Figure 2:
ITGB1 knockdown inhibited osteoblast differentiation and migration. MC3T3-E1 cells were transfected with si-ITGB1. The transfection efficiency of ITGB1 was tested by (a and b) Western blot and (c) qRT-PCR. ALP activity was determined by (d) ALP activity assay and (e and f) ALP staining. Scale bar = 200 μm. (g and h) ARS staining was applied to evaluate the degree of osteoblast matrix mineralization. Scale bar = 200 μm. (i-m) The levels of osteogenic markers (OCN, OPN, ON, and BSP) were measured by Western blot. Osteoblast migration was measured by (n and o) wound healing (Scale bar = 100 μm) and (p and q) transwell assays (Scale bar = 50 μm). ✶P<0.05; ✶✶P<0.01; ✶✶✶P<0.001. n=3. ITGB1: Integrin β1, qRT-PCR: Quantitative real-time polymerase chain reaction, ALP: Alkaline phosphatase, ARS: Alizarin red staining, OCN: Osteocalcin, OPN: Osteopontin, ON: Osteonectin, BSP: Bone sialoprotein, si-NC: Small interfering negative control, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

ITGB1 facilitated the activation of the ERK1/2 signaling pathway

To clarify the molecular mechanism of ITGB1 affecting the differentiation and migration of osteoblasts, p-ERK1/2 and ERK1/2 levels were measured by Western blot. Results revealed that ITGB1 over-expression exhibited an obvious increase in p-ERK1/2 level but no significant change in ERK1/2 level [Figure 3a and b]. However, ITGB1 knockdown showed the opposite effect on p-ERK1/2 level, with an obvious decrease in p-ERK1/2 level [Figure 3c and d]. The inhibitor of the ERK1/2 signaling pathway, U0126, was utilized to block this pathway. Western blot results revealed that p-ERK1/2 level was signally elevated in the ITGB1 over-expression group. Conversely, this level was notably reduced after treatment with U0126 [Figure 3e and f]. These results indicated that ITGB1 activated the ERK1/2 pathway, potentially contributing to its promotion of osteoblast differentiation and migration.

ITGB1 facilitated the activation of the ERK1/2 signaling pathway. The protein levels of p-ERK1/2 and ERK1/2 were measured in MC3T3-E1 cells treated with (a and b) pcDNA3.1-ITGB1, (c and d) si-ITGB1, and (e and f) pcDNA3.1-ITGB1 combined with U0126. ✶✶P<0.01. n=3. ITGB1: Integrin β1, ERK1/2: Extracellular signal-regulated kinase 1/2, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, pcDNA: Plasmid cloning deoxyribonucleic acid, sh-NC: short hairpin-negative control.
Figure 3:
ITGB1 facilitated the activation of the ERK1/2 signaling pathway. The protein levels of p-ERK1/2 and ERK1/2 were measured in MC3T3-E1 cells treated with (a and b) pcDNA3.1-ITGB1, (c and d) si-ITGB1, and (e and f) pcDNA3.1-ITGB1 combined with U0126. ✶✶P<0.01. n=3. ITGB1: Integrin β1, ERK1/2: Extracellular signal-regulated kinase 1/2, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, pcDNA: Plasmid cloning deoxyribonucleic acid, sh-NC: short hairpin-negative control.

Suppression of the ERK1/2 pathway attenuated the effects of ITGB1 on osteoblast differentiation and migration

To elucidate whether the ERK1/2 pathway affected the role of ITGB1 on osteoblast differentiation and migration, U0126 and pcDNA3.1-ITGB1 were transfected into MC3T3-E1 cells to assess osteoblast differentiation and migration. ALP and ARS straining assays revealed that ITGB1 over-expression increased the percentage of positive cells. However, upon treatment with U0126, the positive cells were markedly reduced [Figure 4a-d]. Subsequently, Western blot results revealed that the levels of OCN, OPN, ON, and BSP increased by ITGB1 overexpression were attenuated by U0126 [Figure 4e-i]. Wound-healing results revealed that the wound-healing rate of osteoblast was significantly elevated following transfection with pcDNA3.1-ITGB1. After treatment with U0126, wound healing decreased [Figure 4j and k]. Similarly, transwell assay displayed that osteoblast migration enhanced by pcDNA3.1-ITGB1 dramatically declined following treatment with U0126 [Figure 4l and m]. These findings demonstrated that ITGB1 may influence the differentiation and migration of osteoblasts through the ERK1/2 pathway.

Suppression of the ERK1/2 pathway attenuated the effects of ITGB1 on osteoblast differentiation and migration. U0126 and pcDNA3.1-ITGB1 were transfected into MC3T3-E1 cells. (a and b) ALP activity was determined by ALP staining. Scale bar = 200 μm. (c and d) ARS staining was applied to evaluate the degree of osteoblast matrix mineralization. Scale bar = 200 μm. (e-i) The levels of osteogenic markers (OCN, OPN, ON, and BSP) were measured by Western blot. Osteoblast migration was measured by (j and k) wound healing (Scale bar = 100 μm) and (l and m) transwell assays (Scale bar = 50 μm). ✶P<0.05; ✶✶P<0.01. n=3. ITGB1: Integrin β1, qRT-PCR: Quantitative real-time polymerase chain reaction, ALP: Alkaline phosphatase, ARS: Alizarin red staining, OCN: Osteocalcin, OPN: Osteopontin, ON: Osteonectin, BSP: Bone sialoprotein, ERK1/2: Extracellular signal-regulated kinase 1/2. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, pcDNA: Plasmid cloning deoxyribonucleic acid.
Figure 4:
Suppression of the ERK1/2 pathway attenuated the effects of ITGB1 on osteoblast differentiation and migration. U0126 and pcDNA3.1-ITGB1 were transfected into MC3T3-E1 cells. (a and b) ALP activity was determined by ALP staining. Scale bar = 200 μm. (c and d) ARS staining was applied to evaluate the degree of osteoblast matrix mineralization. Scale bar = 200 μm. (e-i) The levels of osteogenic markers (OCN, OPN, ON, and BSP) were measured by Western blot. Osteoblast migration was measured by (j and k) wound healing (Scale bar = 100 μm) and (l and m) transwell assays (Scale bar = 50 μm). ✶P<0.05; ✶✶P<0.01. n=3. ITGB1: Integrin β1, qRT-PCR: Quantitative real-time polymerase chain reaction, ALP: Alkaline phosphatase, ARS: Alizarin red staining, OCN: Osteocalcin, OPN: Osteopontin, ON: Osteonectin, BSP: Bone sialoprotein, ERK1/2: Extracellular signal-regulated kinase 1/2. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, pcDNA: Plasmid cloning deoxyribonucleic acid.

DISCUSSION

Fractures are a major public health problem worldwide. In addition to morbidity and mortality, fractures result in a high economic cost for health in relation to hospitalization, surgeries, outpatient care, and chronic care.[28] Osteoblasts, the primary functional cells of bone formation, form new bone by migrating to specific sites, differentiating, secreting bone matrix, and promoting matrix mineralization. The features are the reason why osteoblasts are often studied in the study of bone fracture healing. In this current study, we investigated the effects of ITGB1 on osteoblast differentiation and migration, and the results revealed that ITGB1 expression gradually increased during osteoblast differentiation. Moreover, ITGB1 overexpression facilitated the differentiation and migration of osteoblasts, whereas ITGB1 knockdown it. Furthermore, ITGB1 overexpression enhanced the activation of ERK1/2 signaling, thereby promoting osteoblast differentiation and migration.

Integrins are expressed extensively as cell-surface molecules. They play important roles in a variety of biological processes, such as cell migration, growth, differentiation, and apoptosis.[29] ITGB is reportedly the most important integrin receptor subunit of adhesion ligands of osteoblast extracellular matrix such as collagen, fibronectin, and laminin.[30] Another study revealed that ITGB1 silencing blocked osteoblast proliferation and differentiation.[31] Notably, one research has demonstrated that ITGB1 regulates the formation of osteoblasts by mediating adhesion onto bone matrix.[32] In the current study, ITGB1 expression was measured in MC3T3-E1 cells at different incubation times (0, 4, 7, 14, 21, and 28 days) for the 1st time. Our findings revealed that ITGB1 expression gradually increased with increased incubation time. Moreover, ITGB1 overexpression exhibited a more pronounced increase in ALP and ARS positive cells. We further examined markers of osteoblast differentiation (OPN, ON, OCN, and BSP) during osteogenesis[33] and discovered that ITGB1 overexpression enhanced the levels of OPN, ON, OCN, and BSP. ITGB1 overexpression also facilitated osteoblast migration. Conversely, ITGB1 knockdown exhibited the opposite effects on osteoblast differentiation and migration.

The regulation of integrins on cell function is very complex and requires the coordination of multiple signaling pathways.[20] After the integrin binds to the ligand, the signaling molecule is activated to transmit the extracellular signal into the cell, which requires the participation of various proteins, including FAK, Src, and various cytoskeletal proteins.[34] Subsequently, the phosphorylation of these proteins further triggers a cascade of activation of MAPK.[35,36] In addition, ITGB1 can directly activate Shc protein and trigger the cascade reaction of MAPK pathways such as ERK, JNK, and p38.[37] ERK1/2 signaling pathway and p38MAPK signaling pathway are considered to be closely related to the regulation of osteoblast function.[38,39] Previous studies have shown that the activation of ERK/MAPK signals is involved in osteogenic differentiation, promoting BMSCs or osteoblast precursor cells to enter the osteogenic differentiation state.[40-42] In this study, we utilized Western blot to assess the relationship of ITGB1 with the ERK1/2 pathway in MC3T3-E1 cells. Results revealed that ITGB1 overexpression promoted the phosphorylation level of ERK1/2, whereas ITGB1 knockdown inhibited it. This finding suggested that ITGB1 overexpression may facilitate the activation of the ERK1/2 pathway. Subsequently, we conducted a rescue experiment with ERK/MAPK signaling inhibitor to support this conjecture. Results showed that the differentiation and migration of osteoblasts were somewhat restored but cannot completely recover to the level of ITGB1 that was not over-expressed. Therefore, in addition to ERK1/2 signaling, ITGB1 may also regulate osteoblast differentiation and migration through other pathways.

The presence of certain limitations in this study should not be disregarded. First, this study used only one cell line, and multiple cell lines should be used. Second, further animal model investigations are required to explore the impacts of ITGB1 on bone loss after fracture. Third, we focused on the ameliorating effects of ITGB1 on cell models, but its efficacy in patients with bone loss after fracture requires further verification. The current research also has certain flaws in experimental design. For example, only one siRNA was used to knock down ITGB1. Considering siRNA’s off-target effects, at least two siRNAs should be used for the knockdown of ITGB1. Furthermore, an experimental group treated with U0126 alone was lacking. We will try to address these issues in future studies.

SUMMARY

We demonstrated that ITGB1 exerted an important physiological function in osteoblast differentiation and migration through the ERK1/2 pathway in vitro. In clinical practice, ITGB1 expression level may be an important indicator of fracture healing. This work provides new insights into how ITGB1 modulates bone-injury repair by mediating osteoblast differentiation and migration, hinting at potential treatments for bone injury-related diseases.

AVAILABILITY OF DATA AND MATERIALS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ABBREVIATIONS

ALP: Alkaline phosphatase

ARS: Alizarin red staining

BMP: Bone morphogenetic protein

BSP: Bone sialoprotein

ERK1/2: Extracellular signal-regulated kinase ½

ITGB1: Integrin β1

OCN: Osteocalcin

ON: Osteonectin

OPN: Osteopontin

qRT-PCR: Quantitative real-time polymerase chain reaction

si-RNA: Small interfering RNA

STR: Short tandem repeat

AUTHOR CONTRIBUTIONS

LJJ and WJH: Designed the research study; LJJ, LJ, and WJH: Performed the research; LJJ and WJH: Collected and analyzed the data; LJJ, LJ, and WJH; Involved in drafting the manuscript, and all authors have been involved in revising it critically for important intellectual content. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity. All authors give final approval of the version to be published. All authors meet ICMJE authorship requirements.

ACKNOWLEDGMENT

Not applicable.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Ethical approval and consent to participate is not required as this study does not involve animal or human experiments.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

EDITORIAL/PEER REVIEW

To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double-blind model (authors are blinded from reviewers and vice versa) through an automatic online system.

FUNDING: Not applicable.

References

  1. , , . Systemic bone loss after fracture. Clin Rev Bone Miner Metab. 2018;16:116-30.
    [CrossRef] [PubMed] [Google Scholar]
  2. , , , , , , et al. EULAR/EFORT recommendations for management of patients older than 50 years with a fragility fracture and prevention of subsequent fractures. Ann Rheum Dis. 2017;76:802-10.
    [CrossRef] [PubMed] [Google Scholar]
  3. , , . Osteoporosis. Lancet. 2019;393:364-76.
    [CrossRef] [PubMed] [Google Scholar]
  4. , , , . Bone fracture healing: Perspectives according to molecular basis. J Bone Miner Metab. 2021;39:311-31.
    [CrossRef] [PubMed] [Google Scholar]
  5. , , , , . Bone fracture healing within a continuum bone remodelling framework. Comput Methods Biomech Biomed Engin. 2022;25:1040-50.
    [CrossRef] [PubMed] [Google Scholar]
  6. , , , . Advances in pathogenesis and therapeutic strategies for osteoporosis. Pharmacol Ther. 2022;237:108168.
    [CrossRef] [PubMed] [Google Scholar]
  7. , , , , , . From stem cells to bone-forming cells. Int J Mol Sci. 2021;22:3989.
    [CrossRef] [PubMed] [Google Scholar]
  8. , , , . Cell signaling and transcriptional regulation of osteoblast lineage commitment, differentiation, bone formation, and homeostasis. Cell Discov. 2024;10:71.
    [CrossRef] [PubMed] [Google Scholar]
  9. , . The diverse origin of bone-forming osteoblasts. J Bone Miner Res. 2021;36:1432-47.
    [CrossRef] [PubMed] [Google Scholar]
  10. , . HMGB1 promotes bone fracture healing through activation of ERK signaling pathway in a rat tibial fracture model. Kaohsiung J Med Sci. 2019;35:550-8.
    [CrossRef] [PubMed] [Google Scholar]
  11. , , , . Role of wnt signaling and sclerostin in bone and as therapeutic targets in skeletal disorders. Osteoporos Int. 2023;34:213-38.
    [CrossRef] [PubMed] [Google Scholar]
  12. , , , , , , et al. Osthole promotes bone fracture healing through activation of BMP signaling in chondrocytes. Int J Biol Sci. 2017;13:996-1007.
    [CrossRef] [PubMed] [Google Scholar]
  13. , , , , , , et al. Reconstituted HDL-apoE3 promotes endothelial cell migration through ID1 and its downstream kinases ERK1/2, AKT and p38 MAPK. Metabolism. 2022;127:154954.
    [CrossRef] [PubMed] [Google Scholar]
  14. , , , , , , et al. TIM3 activates the ERK1/2 pathway to promote invasion and migration of thyroid tumors. PLoS One. 2024;19:e0297695.
    [CrossRef] [PubMed] [Google Scholar]
  15. . Hypoxia-induced S-phase kinase-interacting protein 2 knockdown repressed the progression of melanoma through extracellular signal-regulated kinase 1/2 pathway. Cytojournal. 2025;22:9.
    [CrossRef] [PubMed] [Google Scholar]
  16. , , , , , . Ganglioside GD1a enhances osteogenesis by activating ERK1/2 in mesenchymal stem cells of Lmna mutant mice. Aging (Albany NY). 2022;14:9445-57.
    [CrossRef] [PubMed] [Google Scholar]
  17. , , , . Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells. J Cell Biochem. 2010;93:741-52.
    [CrossRef] [PubMed] [Google Scholar]
  18. , , , , , . Eucalyptol induces osteoblast differentiation through ERK phosphorylation in vitro and in vivo. J Mol Med (Berl). 2023;101:1083-95.
    [CrossRef] [PubMed] [Google Scholar]
  19. , , , . Ziyuglycoside I upregulates RUNX2 through ERK1/2 in promoting osteoblast differentiation and bone mineralization. Am J Chin Med. 2021;49:883-900.
    [CrossRef] [PubMed] [Google Scholar]
  20. , , , , , , et al. Targeting integrin pathways: Mechanisms and advances in therapy. Signal Transduct Target Ther. 2023;8:1.
    [CrossRef] [PubMed] [Google Scholar]
  21. , . Chapter 22: Structural and signaling functions of integrins. Biochim Biophys Acta Biomembr. 2020;1862:183206.
    [CrossRef] [PubMed] [Google Scholar]
  22. , , . Family-wide analysis of integrin structures predicted by AlphaFold2. bioRxiv 2023 Preprint, doi: 10.1101/2023.05.02.539023
    [CrossRef] [Google Scholar]
  23. , , , , , , et al. Integrin beta1 regulates proliferation, apoptosis, and migration of trophoblasts through activation of phosphoinositide 3 kinase/protein kinase B signaling. J Obstet Gynaecol Res. 2021;47:2406-16.
    [CrossRef] [PubMed] [Google Scholar]
  24. , . Integrin-mediated adhesion and mechanosensing in the mammary gland. Semin Cell Dev Biol. 2021;114:113-25.
    [CrossRef] [PubMed] [Google Scholar]
  25. , , . β1-integrin-a key player in controlling pancreatic beta-cell insulin secretion via interplay with snare proteins. Endocrinology. 2022;164:bqac179.
    [CrossRef] [PubMed] [Google Scholar]
  26. , , , , , , et al. Dissection of cellular communication between human primary osteoblasts and bone marrow mesenchymal stem cells in osteoarthritis at single-cell resolution. Int J Stem Cells. 2023;16:342-55.
    [CrossRef] [PubMed] [Google Scholar]
  27. , , , , , , et al. Insight into the role of integrins and integrins-targeting biomaterials in bone regeneration. Connect Tissue Res. 2024;65:343-63.
    [CrossRef] [PubMed] [Google Scholar]
  28. , , , , , , et al. Traumatic fracture of the pediatric cervical spine: Etiology, epidemiology, concurrent injuries, and an analysis of perioperative outcomes using the kids' inpatient database. Int J Spine Surg. 2019;13:68-78.
    [CrossRef] [PubMed] [Google Scholar]
  29. , . Multiscale models of integrins and cellular adhesions. Curr Opin Struct Biol. 2023;80:102576.
    [CrossRef] [PubMed] [Google Scholar]
  30. , , , , , , et al. Integrin β1 orchestrates the abnormal cell-matrix attachment and invasive behaviour of e-cadherin dysfunctional cells. Gastric Cancer. 2022;25:124-37.
    [CrossRef] [PubMed] [Google Scholar]
  31. , , , , , , et al. Research progress on the regulatory mechanism of integrin-mediated mechanical stress in cells involved in bone metabolism. J Cell Mol Med. 2024;28:e18183.
    [CrossRef] [PubMed] [Google Scholar]
  32. , , , , , , et al. Integrin α2β1 deficiency enhances osteogenesis via BMP-2 signaling for accelerated fracture repair. Bone. 2025;190:117318.
    [CrossRef] [PubMed] [Google Scholar]
  33. , , . Regulation of osteoblast differentiation by cytokine networks. Int J Mol Sci. 2021;22:2851.
    [CrossRef] [PubMed] [Google Scholar]
  34. , , , , , , et al. Obacunone inhibits RANKL/M-CSF-mediated osteoclastogenesis by suppressing integrin-FAK-Src signaling. Cytokine. 2023;164:156134.
    [CrossRef] [PubMed] [Google Scholar]
  35. , , , , . Upregulated desmin/integrin β1/MAPK axis promotes elastic cartilage regeneration with increased ECM mechanical strength. Int J Biol Sci. 2023;19:2740-55.
    [CrossRef] [PubMed] [Google Scholar]
  36. . Signal transduction mechanisms of focal adhesions: Src and FAK-mediated cell response. Front Biosci (Landmark Ed). 2024;29:392.
    [CrossRef] [PubMed] [Google Scholar]
  37. , , , , , , et al. Targeting MAPK/NF-κB pathways in anti-inflammatory potential of rutaecarpine: Impact on Src/FAK-mediated macrophage migration. Int J Mol Sci. 2021;23:92.
    [CrossRef] [PubMed] [Google Scholar]
  38. , , , , . Sika pilose antler type I collagen promotes BMSC differentiation via the ERK1/2 and p38-MAPK signal pathways. Pharm Biol. 2017;55:2196-04.
    [CrossRef] [PubMed] [Google Scholar]
  39. , , , , , , et al. LPS-stimulated inflammation inhibits BMP-9-induced osteoblastic differentiation through crosstalk between BMP/MAPK and smad signaling. Exp Cell Res. 2016;341:54-60.
    [CrossRef] [PubMed] [Google Scholar]
  40. , , , , , , et al. ETV2 promotes osteogenic differentiation of human dental pulp stem cells through the ERK/MAPK and PI3K-Akt signaling pathways. Stem Cell Res Ther. 2022;13:495.
    [CrossRef] [PubMed] [Google Scholar]
  41. , , , , , , et al. Biphasic regulation of osteoblast development via the ERK MAPK-mTOR pathway. Elife. 2022;11:e78069.
    [CrossRef] [PubMed] [Google Scholar]
  42. , , , , , , et al. Nerve growth factor promote osteogenic differentiation of dental pulp stem cells through MEK/ERK signalling pathways. J Cell Mol Med. 2024;28:e18143.
    [CrossRef] [PubMed] [Google Scholar]

Fulltext Views
2,888

PDF downloads
1,707
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles: