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PEDF and Stem Cell Fate

Recently, in 2015, a research group reported that PEDF is a determinant of stem cell fate.  The research group published an overview of our present scientific understanding how PEDF and PEDF peptides regulate stem cells. The review also covered potential clinical applications of PEDF proteins or peptides.

In the same year, a second research group reported that the cytoprotective effects of pigment epithelium-derived factor (PEDF) requires interactions between a region with a distinct ectodomain on the PEDF receptor (PEDF-R). A peptide scanning approach employing custom made synthetic peptides and peptide binding assays was used for studying the interaction of PEDF with its receptor, PEDF-R. It was found that the sequence region composed of positions 98 to 114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects.

These findings added new functions to the already known multiple functions pigment epithelium-derived factor is known to have in mammalian cells.

What is PEDF?

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein widely expressed in multiple organs with broad biological activities. PEDF exhibits multiple functions including antiangiogenic (inhibits the process of new blood vessel formation), antitumor (inhibits the formation or growth of a tumor), anti-inflammatory (reduces inflammation), neurotrophic properties (regulates growth, differentiation, and survival of neurons), cytoprotection and inhibiting oxygen-glucose deprivation (OGD)-induced cardiomyocytes apoptosis, as well as several other functions.


PEDF is a member of the serpin superfamily with non-inhibitory functions. PEDF exhibits neurotrophic, neuro-protective and antiangiogenic properties and is widely expressed in the developing and adult nervous systems. Furthermore, PEDF is important for bone development as well as for the modulation of resident stem cell populations in the brain, muscle, and eye. PEDF also promotes stem cell renewal and is important as a regulator of bone development.

The crystal structure of glycosylated human PEDF was reported in 2001 at a resolution of 2.85 Å.

Figure 1: Structure of human PEDF. The model illustrating the secondary structure is on the left, and the model showing the surface of the protein is on the right. PEDF is an anti-angiogenic and neurite growth-promoting factor.  PDB ID: 1IMV. Mw: ~ 50 kDa; but 44,277 dalton, based on the sequence, pI is estimated at 5.8.
 

Known functions of PEDF and PEDF peptides

  • Determines stem cell fate.
  • Potent inhibitor of angiogenesis in the mammalian ocular compartment (eye).
  • Potent proliferation inhibitor in various cell types.
  • Anti-tumor effect.
  • Increases bone mass.
  • Improves bone plasticity.
  • Regulates wound healing.
  • Acts against a broad range of angiogenic/proliferative inducer molecules possibly through an apoptotic mechanism.
  • Induces a neuronal phenotype in both cultured human retinoblastoma Y79 and Weri cells when added at nanomolar concentrations.
  • Promotes neuronal survival of the cerebellar granule.
  • Promotes both survival and differentiation of developing spinal motor neurons.
  • Prevents death of cerebellar granule neurons.
  • Prevents death of spinal motor neurons.
  • Prevents death of developing primary hippocampal neurons caused by glutamate cytotoxicity.
  • Prevents hydrogen peroxide-induced apoptosis of retinal neurons.
  • Delays death of photoreceptors in the mouse model of retinitis pigmentosa.
  • Supports both normal development of the photoreceptor neurons and opsin expression after removal of the retinal pigment epithelium.
  • Inhibits microglial growth.
  • Modulates different apoptotic pathways.
  • Protects against hypoxia-induced cell death.
  • PEDF production decreases with age and in some diseases, such as nephropathy.
  • Others

Functional epitopes

Functional epitopes have been identified. A PEDF-derived short peptide (PSP) is known to induce satellite cell proliferation and promotes muscle regeneration. Whereas a 44-amino acid peptide fragment of PEDF binds receptors on the surfaces of different neuron types were it determines neurotrophic activity and neuronal differentiation. So far four isoforms of secreted human and bovine PEDF have been observed. The PEDF protein contains 418 amino acids. The first 35 residues at the N-terminus are not structured, and the anti-angiogenic properties and neurotrophic activities are localized in the N-terminal region. The C-terminal region interacts with the membrane receptor. The functional domains of PEDF are listed in Table 1.

Table 1: Functional domains of PEDF (Source: Belkacemi et al. 2016).

Function

Peptide sites

Anti-angiogenesis

34-mer peptide region (residues 24–57).

SPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAA

Collagen binding

(anti-angiogenesis)

Asp256,Asp258, Asp300 (negatively charged), Arg149, Lys166, Lys167 (positively charged).

Asp255, Asp257 and Asp299 are critical to collagen-I-binding.

Cell differentiation

44-mer peptide region (residues 58–101)

VSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTE

Heparin binding

Arg145, Lys146 and Arg148

Hyaluronan

Lys189, Lys191, Arg194 and Lys197 form a motif that is critical for hyaluronan binding.

Laminin binding

34-mer peptide region (residues 44–77).

DPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTN

Phosphorylation

Ser24, Ser114, Ser227

Neurotrophy

44-mer peptide region (residues in humans 78–121)

VLLSPLSVATALSALSLGAEQRTESIIHRALYYDLINNPDIHGT,

ILLSPLSVATALSALSLGAEQRTESVIHRALYYDLINNPDIHST

Tumor cell apoptosis

34-mer peptide region (residues 24–57)

SPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAA



Figure 2: Schematic diagram of PEDF functions. The N-terminal end contains anti-angiogenesis effects. This region has been reported to inhibit Wnt receptor, LRP6, in differentiated cells. The 44-mer and a 20-mer peptide stretch within this region appear to function in neuronal differentiation and muscle progenitor proliferation. The full-length PEDF protein induces mesenchymal stem cells (MSC) to the osteoblast linesage and affect pluripotency of embryonic stem cells (ESC) and apoptosis of inducible pluripotent stem cells.

 

Figure 2: Functional domains of PEDF. Locations for the 34mer peptide and the 44mer peptide are shown in yellow within the structure of human PEDF. The carbohydrate moiety of N-acetyl-D-glucosamine is shown in blue. For the 44mer peptide, the structure is rotated to allow for a better view of the peptide domain.


PEDF-PEDF-R Interactions 

In 2015 Kenealey et al. used a peptide scanning approach for studying the interaction of PEDF with its receptor PEDF-R. The goal was to elucidate the mechanism how PEDF exerts cytoprotection function.  Earlier molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78–121). The use of binding assays demonstrated that PEDF-R binds to the 44-mer peptide.  The peptide P1from the PEDF-Rectodomain was demonstrated to have affinity for the 44-mer and a shorter fragment within it, a 17-mer peptide (positions 98–114). Alanine scanning using small peptide fragments (17-mers) of PEDF revealed key interacting residues responsible for binding to PEDF-R. The 17-mer contains a novel PEDF-R binding region important for retino-protection. Kenealey suggested that altered PEDF peptides could be exploited pharmacologically to improve protection of photoreceptors from degeneration.

Table 2: PEDF peptides used for studying receptor PEDF binding

             (Source: Kenealey et al. 2015).

Name

Sequence

P1

TSIQFNLRNLYRLSKALFPPEPLVLREMCKQGYRDGLRFL

34-mer

FFVPVNKLAAVSNFGYDLYRVRSSMSPTTN

44-mer

VLLSPLSVATALSALSLGADQRTESIIHRALYYDLISSPDIHGT

17-mer

QRTESIIHRALYYDLIS

This peptide still retains affinity to PEDF-R.

Q98A

ARTESIIHRALYYDLIS

R99A

QATESIIHRALYYDLIS

T100A

QRAESIIHRALYYDLIS

E101A

QRTASIIHRALYYDLIS

S102A

QRTEAIIHRALYYDLIS

I103A

QRTESAIHRALYYDLIS

I104A

QRTESIAHRALYYDLIS

H105A

QRTESIIARALYYDLIS

R106A

QRTESIIHAALYYDLIS

L108A

QRTESIIHRAAYYDLIS

Y109A

QRTESIIHRALAYDLIS

Y110A

QRTESIIHRALYADLIS

D111A

QRTESIIHRALYYALIS

L112A

QRTESIIHRALYYDAIS

I113A

QRTESIIHRALYYDLAS

S114A

QRTESIIHRALYYDLIA

 

PEDF Protein Sequence

>1IMV_A Chain A, 2.85 A Crystal Structure Of Pedf
NPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSNFGYDLYRVRSSMSPTTNVLLSPLSVATALS
ALSLGADERTESIIHRALYYDLISSPDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEK
SYGTRPRVLTGNPRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLED
FYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHD
IDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAG
TTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP

   

Osteodystrophy

Osteodystrophy, the defective development of bone, appears to be caused by defects in PEDF expression. Recently, it was shown that null mutations in PEDF, the protein product of the SERPINF1 gene, are the cause of osteogenesis imperfecta (OI) type VI. A PEDF-knockout (KO) mouse exhibited elements similar to the human disease. The result of missing PEDF is diminished bone mineralization and the propensity to bone fracture.

PEDF and mesenchymal stem cell

PEDF directs human mesenchymal stem cell (hMSC) commitment to the osteoblast lineage and modulates Wnt/β-catenin signaling. Wnt/β-catenin is a major regulator of bone development.

PEDF peptides inhibit Wnt/β-catenin signaling and increase mineralization

Belinsky et al. showed that PEDF peptides inhibit Wnt/β-catenin signaling. A 34-mer fragment of PEDF (44–77 aa) and its mutated versions were used for the study (sequence: DPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTN, N-acetylation, and C-amidation). The research group could show that short-term exposure to PEDF peptides or DKK1 antagonizes Wnt signaling in hMSCs. Adding the native PEDF 34-mer and a k→a PEDF 34-mer significantly increased mineralization when added during the last 7 days of the differentiation protocol.

Reference

Belkacemi L, Zhang SX. Anti-tumor effects of pigment epithelium-derived factor (PEDF): implication for cancer therapy. A mini-review. Journal of Experimental & Clinical Cancer Research : CR. 2016;35:4. doi:10.1186/s13046-015-0278-7.

Belinsky GS, Sreekumar B, Andrejecsk JW, et al. Pigment epithelium–derived factor restoration increases bone mass and improves bone plasticity in a model of osteogenesis imperfecta type VI via Wnt3a blockade. The FASEB Journal.
2016;30(8):2837-2848. doi:10.1096/fj.201500027R.

Ho T-C, Chiang Y-P, Chuang C-K, et al. PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells. American Journal of Physiology - Cell Physiology. 2015;309(3):C159-C168. doi:10.1152/ajpcell.00344.2014.

Kenealey J, Subramanian P, Comitato A, et al. Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor. The Journal of Biological Chemistry. 2015;290(42):25241-25253. doi:10.1074/jbc.M115.645846.

Sagheer U, Gong J, Chung C. Pigment Epithelium-Derived Factor (PEDF) is a Determinant of Stem Cell Fate: Lessons from an Ultra-Rare Disease. Journal of developmental biology. 2015;3(4):112-128. doi:10.3390/jdb3040112.

Simonovic M, Gettins PGW, Volz K. Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(20):11131-11135. doi:10.1073/pnas.211268598.

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