Insulin-Like Growth Factor Binding Protein 5: A Novel Regulator of Early Osteogenic Differentiation of hMSCs

Insulin-like growth factor binding protein 5 (IGFBP5) is broadly bioactive, but its role in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) remains to be claried. Herein, we determined that IGFBP5 expression was markedly increased during the early osteogenic differentiation of hMSCs. We then overexpressed and knocked down this gene in hMSCs and evaluated the impact of the manipulation of IGFBP5 expression on osteogenic differentiation based upon functional assays, ALP staining, and the expression of osteogenic markers. Together, these analyses revealed that IGFBP5 overexpression enhanced early osteogenic differentiation as evidenced by increased ALP staining and osteogenic marker induction, whereas knocking down this gene impaired the osteogenic process. Overexpressing IGFBP5 also markedly bolstered extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation level, while IGFBP5 knockdown suppressed this signaling activity. We additionally compared the impact of simultaneous IGFBP5 overexpression and ERK1/2 inhibitor treatment to the effect of IGFBP5 overexpression alone in these hMSCs, revealing that small molecule-mediated EKR1/2 inhibition was sucient to impair osteogenic differentiation in the context of elevated IGFBP5 levels. These ndings indicated that IGFBP5 drives the early osteogenic differentiation of hMSCs via the ERK1/2 signaling pathway. Our results offer value as a foundation for future efforts to study and treat serious bone-related diseases including osteoporosis.


Introduction
Human bone mesenchymal stem cells (hMSCs) are multipotent cells that can self-renew in vivo [1], and that can differentiate into a range of cell types including osteoblasts and adipocytes [2][3][4][5]. When the normal homeostatic balance controlling hMSC differentiation into these two cell types is disrupted such that osteoblastic differentiation is impaired and/or adipogenic differentiation is enhanced, individuals can suffer from signi cant bone loss, contributing to the development of osteoporosis [6]. During osteogenesis, hMSCs upregulate speci c genes in a de ned manner while suppressing the activation of other genes to coordinate phenotypic changes [7], with the early stages of this differentiation process being particularly important as determinants of future cell development [8,9]. In vitro-expanded hMSCs functioning as an optimal model system that can be used to explore the molecular regulation of osteogenesis [10].
Insulin-like growth factor-binding proteins (IGFBPs) are pivotal regulators of the mitogenic activity of Insulin-like growth factors (IGFs) [11], are closely linked to differentiation, proliferation and invasion [12,13]. IGFBP5 is the most highly conserved IGFBP family member among vertebrates, and controls cellular growth, cell fate determination, and tumor cell metastasis [14]. When ovariectomized rats were injected daily with a subcutaneous dose of IGFBP5, this was shown to enhance osteoblast proliferation therein [15]. However, the speci c role of this gene as a regulator of hMSC osteogenesis remains to be clari ed.
Herein, we determined that IGFBP5 is markedly upregulated during the early stages of hMSC osteogenesis, leading us to hypothesize that this IGFBP family member is a key regulator of this differentiation process. Speci cally, we determined that IGFBP5 controls early hMSC osteogenic differentiation via activating the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway.
Together, our data provide a new framework for the understanding of how IGFBP5 can contribute to early hMSC osteogenesis.
Cells were grown until 70% con uent, at which time they were stimulated to undergo osteogenic differentiation by culturing them in media supplemented with 50 mM ascorbic acid, 10 mM βglycerophosphate, and 100 nM dexamethasone (all from Sigma-Aldrich, MO, USA). Fresh differentiation media was added every 3 days.
Following a 10 h incubation at 37°C, media was exchanged and cells were allowed to rest for 72 h. Media was then refreshed and supplemented with 0.5 µg/ml puromycin, which was used to screen cells for 48 h.
Media was then exchanged for fresh puromycine-containing media, and selection was maintained for 6 total days at which time surviving cells had begun proliferating. ALP staining and activity analyses ALP activity was assessed with a staining kit (Beyotime Institute of Biotechnology, Shanghai, China) based on provided directions. Brie y, after two washes with PBS, cells were xed for 20 minutes using 4% formalin. Cells were then incubated two times in ALP buffer (0.1 M NaCl, 0.1 M Tris-hCl, 50 mM MgCl2. 6H2O, pH 9.5) for 5 minutes each, followed by a 30-minute incubation with ALP substrate solution (5 µl BCIP and 10 µl NBT in l ml ALP buffer) at room temperature protected from light. Distilled water was then added to terminate staining, and cells were assessed via microscopy (Olympus, Tokyo, Japan).
An ALP Detection Kit (Nanjing Jiancheng Bioengineering Ltd., Nanjing, China) was additionally used to assess ALP activity based upon provided directions. Brie y, cells were freeze-thawed four times to release endogenous ALP, after which lysates were added to 96-well plates containing ALP substrate and were incubated at 37°C, after which a stop buffer was added to terminate the reaction. The p-nitrophenol product levels in each well were then assessed by analyzing absorbance at 520 nm using a microplate reader (Bio-rad, CA, USA).
qRT-PCR Trizol (Invitrogen) was employed to extract cellular RNA, after which a Reverse Transcription System and Oligo (dT) kit was utilized to prepare cDNA (Thermo Scienti c). For normalization, we utilized β-actin, and primers used for this study are compiled in Table 1. All qRT-PCR reactions were conducted using a SYBR Premix Ex Taq kit (TOYOBO, JAPAN) and a 7500 Real-Time PCR System (ABI, CA, USA), with relative gene expression being evaluated via the 2-ΔΔCT method. Table 1 qRT-PCR primers Gene symbol
Blots were then probed for 1 h using HRP-linked anti-mouse or anti-rabbit IgG (1:5,000; 7076P2 and 7074P2; Cell Signaling Technology). Protein band detection was then conducted with an ECL reagent (BeyoECL Plus; Beyotime Institute of Biotechnology).

Statistical analysis
Data are given as means ± SD, and all experiments were conducted in triplicate. Data were compared via one-way analyses of variance (ANOVAs), with P < 0.05 as the signi cance threshold.

Assessment of IGFBP5 expression during hMSC osteogenesis
We started by evaluating IGFBP5 expression dynamics during osteogenic differentiation of hMSCs, revealing that this gene was gradually upregulated over time until reaching a maximal expression level on day 7 (Fig. 1A). This indicated that IGFBP5 may be a key regulator of early osteogenic differentiation of hMSC.
hMSC transduction At 6 days post-lentiviral transduction, remaining hMSCs were puromycin-resistant, indicating good transduction e ciency. These cells grew effectively and exhibited GFP expression when evaluated via uorescent microscopy (Fig. 1B). The e cacy of lentiviral transduction was additionally con rmed via qRT-PCR and Western blotting ( Fig. 1C and D).

ALP staining and activity
The osteoblastic differentiation of hMSCs was evaluated on days 3 and 7 post-induction via ALP staining. Consistent with positive staining, cells were stained a blue-violet color. Notably, staining intensity was signi cantly greater in IGFBP5-overexpressing cells ( Fig. 2A), whereas it was signi cantly decreased in cells in which IGFBP5 was knocked down (Fig. 2B). This result was also con rmed via quantifying intracellular ALP activity, again revealing that ALP staining intensity was increased in IGFBP5-overexpressing cells and decreased in cells transduced with an IGFBP5-speci c shRNA relative to control cells (Fig. 2C and D).

Modulation of IGFBP5 expression impacts hMSC osteogenesis
In an effort more fully understand the impact of IGFBP5 expression on hMSC osteogenic differentiation, we additionally assessed osteogenic marker gene expression patterns in cells prepared as above. We found that IGFBP5 overexpression markedly enhanced levels of the osteogenic marker genes RUNX2, OCN and ALP at the RNA and protein levels, whereas IGFBP5 knockdown suppressed the induction of both of these genes on days 3 and 7 of the differentiation process (Fig. 2E, F and Fig. 2G, H). IGFBP5 expression impacts ERK1/2 signaling in hMSCs Next, we evaluated the impact of IGFBP5 expression on ERK1/2 signaling in the context of osteogenesis by Western blotting, revealing that both IGFBP5 overexpression and knockdown were linked with increased and decreased p-ERK1/2 levels ( Fig. 3A and B).

Discussion
Osteoporosis is a disease that causes progressive bone loss, increasing the susceptibility of affected patients to bone fractures. The ability of hMSCs to differentiate into osteoblasts, osteocytes, and adipocytes ultimately controls to development of bone and fat tissues [16,17]. Impairment of hMSC osteogenesis can impair bone formation, and such impairment is a common hallmark of osteoporosis [6,18]. It is therefore essential that the molecular mechanisms regulating hMSC hallmark be better understood in order to guide the treatment of osteoporosis and bone fractures.
Herein, we found that IGFBP5 expression in hMSCs increased over time during osteoblastic differentiation, with maximum expression levels being reached on day 7 of this process (Fig. 1A). This showed that IGFBP5 may be a key regulator of the early phases of the osteogenic differentiation process in these cells. To test this possibility, we generated hMSCs in which IGFBP5 was stably overexpressed or knocked down using lentiviral constructs (Fig. 1B-D).
We found that cells overexpressing IGFBP5 exhibited more robust ALP staining and activity, whereas the opposite was true in cells in which this gene was knocked down (Fig. 2A,C; Fig. 2B,D). In line with these ndings, osteogenic marker gene expression was markedly increased during hMSC osteogenesis in cells overexpressing IGFBP5 (Fig. 2E,G), while the expression of these marker genes was suppressed following IGFBP5 knockdown (Fig. 2F,H). These data suggest that IGFBP5 functions as a positive regulator of early hMSC osteoblastogenesis.
Of the six known IGFBP family members, IGFBP5 is the most broadly bioactive and is expressed in many different cells and tissues [13,19,20]. The relationship between IGFBP5 and osteogenic differentiation, however, remains to be fully clari ed. There is some evidence that IGFBP5 can enhance the osteogenic differentiation of umbilical cord stem cells and periodontal ligament stem cells (PDLSCs) [21], and recombinant human IGFBP5 (rhIGFBP5) can promote PDLSC migration, chemotaxis, and osteo/dentinogenic differentiation [22]. However, IGFBP5 overexpression has also been shown to decrease in vitro osteoblastogenesis [12], and there is some evidence that this protein can also restrain skeletal growth [23]. As such, IGFBP5 may play cell-and tissue-speci c roles in regulating physiological activities. As such, in the present study, we speci cally evaluated the impact of IGFBP5 on hMSC osteogenesis.
The mechanistic basis by which IGFBP5 controls osteogenesis has yet to be clari ed. The differentiation of hMSCs into osteoblasts is controlled by the coordinated simultaneous activation of many signaling pathways, making it essential to understand which of these pathways function downstream of IGFBP5. In prior research, IGFBP5 was shown to modulate dental pulp stem cell dentinogenesis by controlling the ERK signaling pathway [21]. Consistent with such activity, IGFBP5 also impacts the growth of pancreatic cancer cells by modulating ERK1/2 signaling [24]. As such, we hypothesized that IGFBP5 may also control osteogenesis via the ERK1/2 signaling pathway. Consistent with this model, we determined that overexpressing or knocking down IGFBP5 was su cient to alter ERK1/2 phosphorylation levels during hMSCs osteogenesis ( Fig. 3A and B). The treatment of these cells with ERK1/2 inhibitors was also su cient to reverse the impact of IGFBP5 overexpression, on hMSC osteogenic differentiation ( Fig. 3C and Fig. 4). These data therefore con rmed that IGFBP5 signals via ERK1/2 in order to control the early osteogenic differentiation of hMSCs.
Together, our data indicate that IGFBP5 serves as a key regulator of early-stage osteoblastogenesis in hMSCs. As a novel positive regulator of this important differentiation process, IGFBP5 may thus be a viable target for future studies of the treatment of osteoporosis and other bone-related diseases.