Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation*

Background: Phlpp1 is a tumor suppressor that represses Akt2 and other signaling pathways. Results: Chondrocyte proliferation, matrix production, Akt2 phosphorylation, and Fgf18/Erk1/2 signaling were increased in Phlpp1−/− mice, but levels of the transcription factor FoxO1 were reduced. Conclusion: Phlpp1 deficiency increases Akt2 activity, which diminishes FoxO1 levels and induces Fgf18 expression to stimulate Erk1/2 activity and chondrocyte proliferation. Significance: Phlpp1 inhibition may promote cartilage regeneration. Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1−/− chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.

Skeletal formation is accomplished in part through the process of endochondral ossification. In this developmental program, a cartilage intermediate first arises to serve as a template for de novo bone formation (reviewed in Ref. 1). Strict control of chondrocyte maturation during the cartilaginous phase and subsequent coupling to bone formation ensures acquisition of optimal bone length, density, and shape. Disruption of this process during embryogenesis causes chondrodysplasias and growth plate injuries during adolescence can cause limb shortening (2,3). Cartilage-specific genetic deficiency of anabolic factors can also result in decreased bone density, highlighting the importance of growth plate cartilage coupling to bone for-mation (4 -6). The endochondral ossification process is reinitiated in adults during fracture healing, heterotopic ossification, and osteoarthritic progression (7).
A number of growth factors and cytokines (e.g. BMPs, Fgfs, Igf1, Hhs, Tgf␤) contribute to the formation and maturation of the cartilaginous phase of endochondral ossification (1). Many of these morphogens directly or indirectly activate Akt (protein kinase B) isoforms that are essential for chondrocyte differentiation (8 -16). Akt activity is controlled by phosphorylation and substrate interactions via scaffolding proteins. A number of phosphatases, including PH-domain leucine-rich repeat protein phosphatases (Phlpp) 2 1/2, dampen Akt activity to promote apoptosis and inhibit cell proliferation (17)(18)(19). Genetic deletion of Phlpp1 (also known as Phlpp, Scop, Plekhe1) slightly reduced snout-to-tail body lengths, but the effects of Phlpp1 on chondrocytes or growth plate structure were not studied (20).
We previously demonstrated that Hdac3 represses Phlpp1 transcription and that Phlpp1 is expressed at higher levels in Hdac3 suppressed chondrocytes (21). Here we examined the effects of Phlpp1 deficiency on skeletal development. Phlpp1 Ϫ/Ϫ mice show increased chondrocyte proliferation and matrix production. Molecular analyses demonstrated Phlpp1 deficiency increased phosphorylation of Akt2, Fgfr3, Mek1/2, and Erk1/2. The elevation in Erk1/2 activity was attributed to increased production of Fgf18, as blocking Fgf receptor (Fgfr)mediated signaling decreased Erk1/2 phosphorylation. Furthermore, Fgfr inhibition suppressed chondrocyte proliferation induced by Phlpp1 deficiency. We conclude that Phlpp1 deficiency promotes chondrocyte differentiation via multiple pathways.

Materials and Methods
Phlpp1-deficient Mice-Phlpp1 Ϫ/Ϫ (20) and wild type (WT) littermates were maintained in an accredited facility with 12-hour light/dark cycles and supplied water and food (Pico-Lab Rodent Diet 20, LabDiet) ad libitum. All animal research was conducted according to National Institutes of Health and the Institute of Laboratory Animal Resources, National Research Council guidelines. The Mayo Clinic Institutional Animal Care and Use Committee preapproved all animal studies. For BrdU labeling studies, mice (n ϭ 5) were injected when they were 5 days old (P5) with 50 g of 5-bromo-2Ј-deoxyuridine (BrdU) labeling reagent (Invitrogen) per kg body weight 2 h prior to euthanasia.
Microcomputed Tomography-Bone volume density in the secondary spongiosa of female distal femurs was measured with ex vivo micro-computed tomography (CT) imaging (CT35; Scanco Medical AG, Basserdorf, Switzerland). Trabecular bone scans were performed at 7 m voxels with an energy setting of 70 kVp and a 300 ms integration time on 8-week-old Phlpp1 Ϫ/Ϫ (n ϭ 4) or WT (n ϭ 3) mice.
Isolation and Culture of Primary Immature Murine Chondrocytes-Primary chondrocytes were collected from P5 Phlpp1 Ϫ/Ϫ or WT mice as previously described (22). Briefly, cartilage from the distal femur and proximal tibia were digested in 3 mg/ml collagenase for 1 h and then overnight in 0.5 mg/ml collagenase. The resulting cell suspension was plated in micromasses consisting of 2 ϫ 10 5 cells in a 10-l drop of DMEM. After 1 h, each well was flooded with DMEM supplemented with 5% FBS, 1% antimycotic/antibiotic for the indicated periods of time.
Alcian Blue Staining-Micromass cultures were fixed with 10% neutral buffered formalin and stained with Alcian blue for 2 h. Tibias from P5 or 4-week-old mice were fixed in 10% neutral buffered formalin, decalcified in 15% EDTA for 7 days, paraffin embedded, sectioned, and stained with Alcian blue.
RNA Isolation and Real-time PCR-Total RNA was extracted from chondrocyte cultures using TRIzol (Invitrogen) and chloroform. Messenger RNA (2 g) was reverse transcribed using the SuperScript III first-strand synthesis system (Invitrogen). The resulting cDNAs were used to assay gene expression via real-time PCR using the following gene-specific primers: Phlpp1 Regulates Chondrocyte Proliferation JUNE 26, 2015 • VOLUME 290 • NUMBER 26 (24). Fold changes in gene expression for each sample were calculated using the 2 Ϫ⌬⌬Ct method relative to control after normalization of gene-specific Ct values to Gapdh Ct values. Each experiment was performed in triplicate and repeated at least three times. The average of these experiments is reported.
Image Quantitation-Images were digitally scanned or collected using phase contrast microscopy. Densitometric values were obtained after converting each image to gray scale and measuring the mean gray values of each condition using Image J software.
Statistical Analysis-Data obtained are the means Ϯ S.E. p values were determined with the Student's t test.

Results
Phlpp1 Deficiency Reduces Bone Length and Density-Phlpp1deficient mice were previously reported to have slightly decreased snout-to-tail lengths and decreased body weight compared with their WT littermates (20). We confirmed shorter femur lengths (Fig. 1A) and further found a decrease in bone mineral density. Micro-CT analyses revealed that Phlpp1 deficiency decreased bone volume density by 23% at 8 weeks of age ( Fig. 1, B, G, H). This was associated with a decrease in trabecular number (Tb.N) (Fig. 1C). Decreased bone mineral density was still apparent in mice at 12 weeks of age (data not shown). Trabecular thickness and trabecular spacing were not altered (Fig. 1, D and E). A trend toward
Phlpp1 Suppresses Chondrocyte Proliferation and Matrix Production-To better understand how Phlpp1 deficiency might stunt bone lengthening and endochondral bone formation, we examined the growth plates of young mice. Tibial growth plates from Phlpp1 Ϫ/Ϫ mice had 50% more cells per proliferative zone area as well as more intense Alcian blue staining (Fig. 2, A and B). BrdU labeling experiments revealed an increase in the number of BrdU-positive cells in Phlpp1-null mice (Fig. 2C). The number of Ki67-positive cells was also enhanced in Phlpp1 Ϫ/Ϫ tibias (data not shown). Thus, despite shorter lengths and reduced endochondral ossification, growth plate chondrocyte proliferation, and matrix content appear to be increased in Phlpp1-deficient long bones.
Phlpp1 Deficiency Increases Fgf18 Production and Activates Fgfr3, Mek, and Erk1/2 Signaling-To determine how Phlpp1 affects chondrocytes, we surveyed a panel of morphogens known to regulate cartilage development (Fig. 4A). Several cytokines (including Tgf␤2/3 and Wnt2b) were modestly higher in Phlpp1 Ϫ/Ϫ cells, but the most highly induced gene of those tested was Fgf18 (Fig. 4A). Fgf18 binds to Fgfrs. To determine if
FoxO1 Represses Fgf18 Expression in Chondrocytes-FoxO1 represses Fgf18 transcription (36) and therefore may link enhanced Akt2 signaling to Erk1/2 activation in Phlpp1 Ϫ/Ϫ chondrocytes. To test this hypothesis, a Phlpp1 deletion mutant (Phlpp1⌬C) that inhibits interactions between Akt2 and Phlpp1 was introduced in chondrogenic ATDC5 cells (18). Phlpp1⌬C promoted Fgf18 production (Fig. 5). This induction was blocked by expression of a constitutively active form of FoxO1 (FoxO1A3, Fig. 5). Furthermore, DNA binding of FoxO1 was required to repress Fgf18 expression as a FoxO1 mutant construct lacking the DNA-binding domain (FoxO1A3HR) FIGURE 4. Phlpp1 deficiency increases Fgf18 expression and Erk1/2 signaling. A-C, Chondrocyte micromass cultures from WT or Phlpp1 Ϫ/Ϫ mice were cultured for 3 days. A, expression levels of indicated morphogens in Phlpp1 Ϫ/Ϫ cells relative to control WT cells were assayed by real-time PCR. The control value was set to 1. *, p Ͻ 0.1; **, p Ͻ 0.05 compared with control. B, immature chondrocytes from WT or Phlpp1 Ϫ/Ϫ mice were cultured in the presence of 100 nM PD173074. Western blotting was performed with the indicated antibodies and (C) the average gray value of the normalized phospho-Erk1/2 signal was determined (n ϭ 3, normalization against total Erk1/2). D and E, immature chondrocytes from WT or Phlpp1 Ϫ/Ϫ mice were cultured in monolayer in the presence of the indicated concentrations of the Fgfr inhibitor (PD173074), the Mek1/2 inhibitor (U0126) or vehicle control (DMSO) for 4 days. MTS assays were performed. These experiments were repeated three times. Data from a representative experiment are shown. *, p Ͻ 0.05 compared with WT vehicle-treated cells; # , p Ͻ 0.05 compared with Phlpp1 Ϫ/Ϫ vehicle-treated cells.

Discussion
Because Phlpp1 deficiency causes growth retardation, we sought to determine if Phlpp1 regulates chondrocyte function during endochondral ossification. Our results show that Phlpp1 is a regulator of endochondral bone development, as chondrocyte proliferation and matrix synthesis are enhanced in Phlpp1-deficient mice. This likely occurs due to the ability of Phlpp1 to directly control the activity of anabolic signaling pathways, including Akt2, PKC, and p70 S6 kinase, that are known facilitators of proliferation and matrix production by chondrocytes (21,37,38). Activation of these pathways triggers subsequent events, including but not limited to, Fgf18 production and downstream activation of Fgfr and Mek/Erk signaling. Thus, Phlpp1 is active during endochondral bone formation. The effects of Phlpp1 deletion are similar to those observed in mice lacking GDF5 or HIF1␣ that are characterized by increased chondrocyte proliferation and decreased long bone length (39,40). The effects of Phlpp1 deficiency on distinct chondrocytes populations, osteoblasts and/or osteoclasts in the developing bone cannot be addressed with this model. Targeted and regulated deletion of Phlpp1 in these and other cell types are needed to fully understand why germline Phlpp1 deficiency produces shorter and thinner bones despite increased BrdU labeling and matrix production.

Phlpp1 Regulates Chondrocyte Proliferation
We also noted that Phlpp1 deficiency increases expression of several growth factors, including robust induction of Fgf18. We also demonstrated enhanced Fgf18 production and signaling through Fgfr leading to enhanced Mek/Erk activation and chondrocyte proliferation (Fig. 7). The effects on increased growth factor expression on matrix production, however, were not explored here. Fgf18 is naturally expressed within joints, the perichondrium and growth plates of developing bones (42,43). Our results are inconsistent with two main observations: 1) that germline Fgf18 deficiency increases chondrocyte proliferation during embryonic development (42,43), and 2) that Fgfr3 activation decreases chondrocyte proliferation and causes limb shortening in humans (44). However, other studies demonstrate that Fgf18 promotes chondrocyte proliferation and cartilage maintenance, particularly of articular chondrocytes (32,43,(45)(46)(47)(48)(49). Moreover, gain-of-function Fgfr3 mutations expand the pool of proliferative, Sox9-positive chondrocytes and decrease chondrocyte hypertrophy (50,51). Fgf18 also promotes chondrogenesis, chondrocyte proliferation and cartilage regeneration in surgically induced models of osteoarthritis (31,32,43,(52)(53)(54)(55)(56). In clinical trials, intra-articular injections of recombinant Fgf18 resulted in dose-dependent increases in total cartilage content, even though thickness of the central medial femorotibial compartment cartilage was unchanged (57). Thus, the effects of Fgf18/Fgfr signaling are complex and may be temporally and spatially controlled. Here we show that increased Fgf18/Fgfr signaling is responsible for some of the effects of Phlpp1 deficiency in micromass cultures. However, the role of Fgf18 within the expanded proliferative zone of the Phlpp1 Ϫ/Ϫ growth plate remains to be determined. Given that Phlpp1 loss of function, either via genetic deficiency or small molecule inhibition, promotes chondrocyte maturation and Fgf18 production in vitro, Phlpp1 is an enticing target for cartilage tissue engineering.
Other groups demonstrated the importance of Akt activity in controlling Fgf18 production and Mek/Erk activation. Conditional deletion of PTEN, a phosphatase that suppresses PI3K/ Akt signaling upstream of Phlpp1, in limb progenitor cells enhanced Fgf18 expression (36). The authors attributed this regulation to decreased levels of FoxO1, which represses Fgf18 promoter activity (36). We also found that disrupting the substrate binding ability of Phlpp1 enhances Fgf18 expression, and that this effect is antagonized by expression of an Akt-insensitive FoxO1 mutant.
In summary, we show that germline deletion of Phlpp1 disrupts limb development, but promotes chondrogenesis and increases cartilaginous matrix production, which may be attributed to increases in cartilage anabolic signaling pathways including Akt2, PKC, and p70 S6 kinase, and relief of FoxO1 suppression of Fgf18. Elevated Fgf18 levels signal through Fgfr to activate Mek/Erk signaling and proliferation. Importantly, Phlpp1 chemical inhibition enhances signaling pathway activation, Fgf18 expression and cartilage matrix production. Thus, Phlpp1 may serve as a target to promote cartilage maintenance and/or regeneration and prevent hypertrophic ossification.