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. Author manuscript; available in PMC: 2011 Jul 18.
Published in final edited form as: J Invest Dermatol. 2009 Aug 27;130(2):398–404. doi: 10.1038/jid.2009.259

BMP signalling induces cell-type specific changes in gene expression programs of human keratinocytes and fibroblasts

Michael Y Fessing 1, Ruzanna Atoyan 3, Ben Shander 3, Andrei N Mardaryev, Vladimir V Botchkarev Jr 4, Krzysztof Poterlowicz 2, Yonghong Peng 2, Tatiana Efimova 5, Vladimir A Botchkarev 1,2,*
PMCID: PMC3138418  NIHMSID: NIHMS302246  PMID: 19710687

Abstract

BMP signalling plays a crucial role in skin development and homeostasis, whereas molecular mechanisms underlying its involvement in regulating gene expression programs in keratinocytes and fibroblasts remain largely unknown. We demonstrate here that several BMP ligands, all BMP receptors and BMP-associated Smad1/5/8 are expressed in human primary epidermal keratinocytes and dermal fibroblasts. Treatment of both cell types by BMP-4 resulted in the activation of the BMP-Smad, but not BMP-MAPK pathways. Global microarray analysis revealed that BMP-4 treatment induces distinct and cell-type specific changes in gene expression programmes in keratinocytes and fibroblasts, which are far more complex versus the effects of BMPs on cell proliferation/differentiation described previously. Furthermore, our data suggest that potential modulation of cell adhesion, extracellular matrix remodelling, motility, metabolism, signalling and transcription by BMP-4 in keratinocytes and fibroblasts is likely to be achieved by the distinct and cell type-specific sets of molecules. Thus, these data provide an important basis for delineating mechanisms that underlie the distinct effects of the BMP pathway on different cell populations in the skin and will be helpful in further establishing molecular signalling networks regulating skin homeostasis in health and disease.

Introduction

Bone morphogenetic protein (BMP) signalling plays important roles in regulating multiple functions in developing and postnatal skin including the control of cellular proliferation and differentiation, epithelial-mesenchymal interactions, melanogenic activity and tissue remodelling (reviewed in (Botchkarev, 2003b)). BMPs are also involved in the controlling the variety of pathobiological processes in the skin including the wound healing, psoriasis, and carcinogenesis (Blessing, 1995; Blessing et al., 1996; Kaiser et al., 1998) Data obtained during the last decade revealed that BMP signalling is capable of modulating the activity of major cellular components of the skin including epidermal and hair follicle keratinocytes, dermal fibroblasts, melanocytes and epithelial stem cells (Andl et al., 2004; D'Souza et al., 2001a; Kobielak et al., 2003; Kobielak et al., 2007; Park and Morasso, 2002; Plikus et al., 2004; Sharov et al., 2005; Sharov et al., 2003; Zhang et al., 2006). However, despite its broad involvement in the control of skin homeostasis and remodelling, the mechanisms of regulation and transcriptional targets for BMP signalling in distinct skin cell populations remain to be elucidated.

BMPs belong to the transforming growth factor-beta (TGF-β) superfamily and bind to the trans-membrane receptor complex formed by the type I (ActR-I/Alk-2, BMPR-IA/Alk-3, BMPR-IB/Alk-6) and type II (BMPR-II, ActR-IIA, ActR-IIB) receptors (Kitisin et al., 2007; Miyazono et al., 2005). Ligand binding to the BMP receptor complex results in phosphorylation of the intracellular domain of the type I receptor by the type II receptor kinase domain and leads to transmission of a signal through the BMP-Smad and/or BMP-MAPK pathways (Kitisin et al., 2007; Miyazono et al., 2005). BMP-specific Smad proteins (Smad1/5/8 or receptor-activated Smads, R-Smads) after phosphorylation form heteromeric complex with Smad4 (or common-partner Smad, Co-Smad) and translocate to the nucleus to regulate the transcription of the BMP-responsive genes. The BMP-MAPK pathway could also employ several key MAP kinases (p38, JNK and/or Erk1/2) (Nohe et al., 2004), while the mechanisms and biological effects of this pathway in cells are much less understood.

Epidermal keratinocytes and dermal fibroblasts are major cellular components of the skin, and their interactions are critical for proper control of skin development and postnatal homeostasis (Fuchs, 2007). BMP signalling serves as powerful regulator of epithelial-mesenchymal interactions in developing skin and is also capable of modulating functional activity of keratinocytes and fibroblasts postnatally (Botchkarev, 2003; Botchkarev and Kishimoto, 2003). However, effects of the BMPs on cellular targets strongly depend on the levels and activity of extracellular BMP antagonists including Noggin that inhibits ligand binding to the BMP receptor complex and modulate a magnitude of BMP signalling (Balemans and Van Hul, 2002).

Data obtained from cultured mouse keratinocytes show that cell infection with BMP-6 expressing adenovirus (Drozdoff et al., 1994) or their treatment with recombinant BMP-2 or BMP-6 proteins under distinct culture conditions led to induction of keratinocyte differentiation (D'Souza et al., 2001b; McDonnell et al., 2001; Park and Morasso, 2002) Thus, in differentiating mouse epidermal keratinocytes, BMP6 stimulated keratin 1 and involucrin expression through BMP-Smad pathway (D'Souza et al., 2001b; McDonnell et al., 2001), and BMP2 activated the expression of Dlx-3 transcription factor (Park and Morasso, 2002). However, stimulatory effects of BMP-2/6 on differentiation process in human epidermal keratinocytes is seen only in the absence of EGF in culture medium as EGF purportedly exerts an inhibitory effect on the BMP pathway (Gosselet et al., 2007).

Data obtained from BMP-6 transgenic mice (promoter: keratin 10) demonstrate that high levels of BMP-6 in the epidermis inhibit epidermal proliferation, while moderate level of the transgene expression stimulated epidermal proliferation, indicating that biological effect of the BMP signalling depends on the intensity of the signal (Blessing et al., 1996). This is consistent with data obtained from Noggin transgenic mice (promoter: keratin 5) that show increased epidermal proliferation and decreased expression of keratinocyte differentiation markers (Sharov et al., 2003).

In contrast to keratinocytes, the effects of BMP signalling on dermal fibroblasts are only poorly understood. BMP-2 treatment of human dermal fibroblasts in 3D culture leads to their chondrogenic differentiation (Zhou et al., 2004). Noggin overexpression in the epidermal keratinocytes is accompanied by thickening of the dermis suggesting the effects of the BMPs on fibroblast collagen-producing activity (Sharov et al., 2003). Constitutive deletion of Noggin in mice is accompanied by increase of p75 kD neurotrophin receptor expression in population of dermal fibroblasts that form the follicular papilla (Botchkarev et al., 1999), in which BMP signalling is important for maintenance of hair-inductive properties (Rendl et al., 2008).

Global microarray data reveal that BMP signalling is involved in regulating the expression of over 500 genes in distinct cell types outside of the skin (Balint et al., 2003; Korchynskyi et al., 2003; Locklin et al., 2001; Miyoshi et al., 2008; Vaes et al., 2002) However, complex effects of the BMP signalling on epidermal keratinocytes and dermal fibroblasts remain unknown. By using global microarray analysis, we show here that BMP signalling causes distinct and cell type-specific changes in gene expression programmes in human primary epidermal keratinocytes and dermal fibroblasts, thus suggesting its involvement in complex regulation of cell adhesion, extracellular matrix remodelling, cellular motility, metabolism, signalling and transcription in both cell types.

Results and Discussion

Components of the BMP signalling pathway are expressed in human keratinocytes and fibroblasts

First, we analyzed expression of the transcripts for distinct components of the BMP signalling pathway in primary human epidermal keratinocytes and dermal fibroblasts. Under normal culture conditions, both cell types expressed all sub-types of the BMP and activin receptors (BMPR-IA/Alk-3, BMPR-IB/Alk-6, BMPR-II, ActR-I/Alk-2, ActR-IIA and ActR-IIB), as well as at least several BMP receptor ligands including BMP-2/4/6 (Fig. 1). These results were consistent with data published previously (Hwang et al., 2001) and suggested that human epidermal keratinocytes and dermal fibroblasts serve as sources and targets of BMP signalling.

Figure 1. Ligand and receptors of the BMP signalling pathway are expressed in human primary epidermal keratinocytes and dermal fibroblasts.

Figure 1

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RNA was isolated from the human keratinocytes and fibroblasts and analysed by semi-quantitative RT-PCR using primers specific for indicated genes. Representative examples of three to four experiments are shown. RNA dilution without reverse transcription was used as a negative control.

BMP-4 treatment leads to activation of the BMP- Smad pathway in human epidermal keratinocytes and dermal fibroblasts

Ligand binding to the BMP receptor complex results in activation of signalling through the SMAD and/or MAP kinase pathways depending on cell type and conditions (Miyazono et al., 2005). Binding of the BMP ligands to preformed type I and type II receptor heteromeric complexes leads to activation of SMAD signalling, while ligand binding to homomeric type I or type II receptor followed by formation of a heteromeric receptor complex likely to result in activation of the BMP-MAPK pathway (Nohe et al., 2002; Nohe et al., 2004). Treatment of keratinocytes and fibroblasts with BMP-4 for different duration of time resulted in increase in Smad1/5/8 phosphorylation in both cell types (Fig. 2).

Figure 2. BMP signalling results in activation of the BMP-SMAD, but not BMP-MAPK pathways in human keratinocytes and fibroblasts.

Figure 2

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Epidermal keratinocytes (A) and dermal fibroblasts (B) were treated with 50 ng/ml recombinant BMP-4 or vehicle control, the cell lysates were prepared at different time-points and analysed by immuno-blotting using antibodies against phospho-Smad1/5/8, phospho-p38 MAPK, phospho ERK1/2, and phosphor-JNK. Representative examples of three to four experiments are shown.

As shown in Fig. 2, cultured keratinocytes and fibroblasts exhibited differential profiles of constitutive activity of the three MAP kinases (p38, Erk1/2 and JNK) under basal unstimulated conditions. Despite the differences in the endogenous levels of phospho-MAP kinases between keratinocytes and fibroblasts, BMP-4 treatment did not cause any significant alterations in their constitutive phosphorylation levels in either cell type (Fig. 2), as well as in the levels of the corresponding total proteins (data not shown). MAP kinase pathways are activated by a number of growth factors including the components of EGF and TGF-beta signalling (Eckert et al., 2002). Interactions between the BMP and MAPK pathways are complex, and active MAPK pathway is capable of inhibiting BMP-Smad signalling through the phosphorylation of the linker region of R-SMADs, prevention of their interaction with SMAD4 and translocation into the nucleus (Kretzschmar et al., 1997). Our data demonstrated that despite constitutive MAPK signalling, BMP-4 treatment of epidermal keratinocytes and dermal fibroblasts activated the BMP-Smad pathway in both cell types (Fig. 2).

BMP-4 treatment differentially affects gene expression programmes in human epidermal keratinocytes and dermal fibroblasts

To define changes in gene expression programmes triggered by the BMP-4 treatment in human epidermal keratinocytes and dermal fibroblasts, RNA samples were processed for global microarray analysis using Affymetrix Human Genome platform, as described previously (Mammucari et al., 2005). RNA was isolated from the cells treated with recombinant BMP-4 or vehicle control for 8 hours to reveal genes that form the early and intermediate responses to BMP-4 stimulation. The micro-array data have been deposited in NCBI's Gene Expression Omnibus (Edgar at al., 2002) and are accessible through GEO Series accession number GSE16111. Gene expression was considered significantly altered if changes in normalized hybridisation signal was 1.8 fold or greater versus the corresponding controls. The alterations in expression levels of selected genes were confirmed by quantitative RT-PCR, and RT-PCR results were generally consistent with data of microarray analysis (Fig. 3 A–C).

Figure 3. BMP signalling causes distinct changes in gene expression programmes in human keratinocytes and fibroblasts.

Figure 3

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Epidermal keratinocytes and dermal fibroblasts were treated with 50 ng/ml of recombinant BMP-4 or vehicle control for 8 hours, the RNA was isolated from the three to four experiments, pooled and gene expression changes were analysed using microarray technology with validation of the data for selected genes using quantitative RT-PCR.

A - qRT-PCR data for selected genes similarly regulated by the BMP signalling both in keratinicytes and fibroblasts.

B - qRT-PCR data for selected genes affected by the BMP signalling only in the keratinicytes but not in the fibroblasts.

C - qRT-PCR data for selected genes affected by the BMP signalling only in the keratinicytes but not in the fibroblasts.

D – F: Functional distribution of the genes similarly regulated by the BMP-4 signalling in keratinocytes and fibroblasts (D), affected by the BMP-4 signalling only in keratinocytes but not in fibroblasts (E) and affected by the BMP-4 signalling only in fibroblasts but not in keratinocytes (F).

Microarray analysis revealed that after BMP-4 treatment, the expression of only 17 genes showed similar changes in expression levels in both keratinocytes and fibroblasts (Suppl. Table 1). This group of genes included well-known BMP targets (Miyazono et al., 2005), such as inhibitors of DNA binding (ID1/2/3/4), components of the BMP pathway (SMAD6/7) and DLX2 transcription factor (Fig. 3 A and Suppl. Table 1). In addition, several other genes including tropomyosin 1 alpha (TPM1) were upregulated in both keratinocytes and fibroblasts after BMP-4 treatment (Suppl. Table 1).

However, expression of the vast majority of genes showed cell-type specific dynamics after BMP-4 treatment and was changed only in one cell type, while remaining un-altered in the other cell type (keratinocytes – 166 genes, fibroblasts – 229 genes, respectively; Suppl. Tables 2 and 3). Large number of these genes were expressed at comparable levels in both cell types, whereas a certain portion of genes (about 39% in keratinocytes and about 22% in fibroblasts) expression of which was altered by BMP-4 in one cell type showed only very low expression levels in the other cell type (Suppl. Tables 2 and 3). This observation suggested that differential effects of the BMP signalling on gene expression in distinct cell types may depend on a number of mechanisms including those controlling the basal expression levels of the BMP target genes.

Genes the expression of which was changed after BMP-4 treatment in a cell type-specific manner belonged to distinct functional classes including those encoding cell adhesion/extracellular matrix proteins, metabolic and proteolytic enzymes, components of the cytoskeleton, and molecules involved in cellular signalling and transcription (Fig. 3 D–F, Suppl. Tables 2, 3). These results demonstrated the complex effects of BMP-4 on gene expression programs in both keratinocytes and fibroblasts, which is consistent with previous observations demonstrating similar effects of the BMPs on other cell types (Balint et al., 2003; Korchynskyi et al., 2003; Locklin et al., 2001; Miyoshi et al., 2008; Vaes et al., 2002). However, our data suggested that a modulation of gene expression programmes by BMP-4 in keratinocytes and fibroblasts is achieved by recruitment of the distinct sets of molecules, which appears to be specific for each cell type.

Interestingly, BMP-4 treatment did not cause changes of proliferative activity in either keratinocytes or fibroblasts, nor did alter the expression of cyclin-dependent kinase inhibitors, such as p21Cip1 and p27Kip1, described previously as TGF-beta/BMP targets that promote cell differentiation (Pardali et al., 2005; Sharov et al., 2006) (data not shown). Under our culture conditions, BMP-4 treatment of primary keratinocytes did not affect the expression of differentiation markers, such as involucrin, loricrin or suprabasal keratins (data not shown). We assume that the presence of other growth modulators, i.e. EGF in culture media may affect the BMP-4 capacity to significantly influence proliferation/differentiation in our models.

Indeed, this hypothesis is consistent with data published previously and demonstrating lack of the effects of BMP-2/6 on the differentiation process of primary epidermal keratinocytes in the presence of EGF in the culture medium (Gosselet et al., 2007). It has been proposed that EGF prevents BMP-mediated keratinocyte differentiation through inhibiting BMP signalling (Gosselet et al., 2007). Notably, our data demonstrated that EGF presence in the culture medium did not preclude BMP signalling via SMAD1/5/8 phosphorylation in either cell type. Yet, our results do not rule out a possibility that EGF-mediated constitutive activity of MAP kinases may exert a negative impact on BMP/SMAD signalling as recently reported (Gosselet et al., 2007).

Interestingly, BMP-4 treatment of primary keratinocytes resulted in marked downregulation of expression of thrombospondin 2 (THBS2) (Suppl. Table 2, Fig. 3 B), which serves as a potent inhibitor of angiogenesis (Lawler and Detmar, 2004). These data are consistent with reports that show stimulatory effects of BMPs on angiogenesis in different models (Araya et al., 2008; Heinke et al., 2008; Liu et al., 2007). Our data, in addition to previously proposed mechanisms, suggest that BMP signalling may stimulate angiogenesis, at least in part, via inhibition of the thrombospondin 2 expression.

Data presented here are also consistent with previous observations showing the involvement of BMP signalling in regulation of extracellular matrix remodelling and activity of matrix metalloproteinases (Rothhammer et al., 2008). However, we show that BMP-4 treatment of keratinocytes and fibroblasts results in modulating the expression of cell type-specific sets of proteolytic enzymes and their inhibitors. In keratinocytes, BMP-4 treatment results in increase of expression of two members of the MMP family (MMP3 and MMP10), while in fibroblasts BMP-4 stimulates expression of the ADAMS family of proteinases (ADAMTS5 and ADAM12; Supplementary Tables 2 and 3, Fig. 3 B,C). Since MMP and ADAMS subfamilies of metalloproteinases have different extracellular targets for cleavage and/or degradation (Malemud, 2006), distinct effects of the BMP-4 on their expression in keratinocytes and fibroblasts may reflect distinct requirement of these molecules for modulating tissue-specific microenvironment of these cells.

BMP-4 treatment also induced cell-type specific changes in expression of the distinct components of the Wnt, NF-kappa B, IGF, FGF, Notch pathways suggesting involvement of the BMP pathway in modulating regulatory signalling networks in keratinocytes and fibroblasts (Suppl. Tables 2 and 3). Interestingly, BMP-4 induced significant cell-type specific change in expression of distinct component of the BMP pathway: BMP-6 expression in keratinocytes (Fig. 4 B; Suppl. Tables 2). These data suggested existence of positive regulatory loop in the BMP signalling in this cell type in addition to negative feedback loops characterized previously (Gazzerro and Canalis, 2006; Kretzschmar et al., 1997; Miyazono et al., 2005).

Our data demonstrating distinct responses to BMP-4 stimulation in keratinocytes and fibroblasts raise a question about mechanisms that determine cell type-dependent specificity in the effects of BMP signalling on the expression of its target genes. Most likely, such cell-type dependent contexts are determined by many factors, a combination of which results in the distinct effects of BMP pathway on gene expression programs in keratinocytes versus fibroblasts. In particular, it has been demonstrated that distinct BMP target genes may require different threshold of the signal to trigger changes in their expression levels (Dosch et al., 1997), and such signalling thresholds could be different in distinct cell types.

Furthermore, presence or absence of distinct co-regulators of gene expression and/or distinct patterns of the promoter (s)/enhancer (s) of chromatin methylation/acetylation may also underlie cell-type specific differences in the effects of BMP signalling on keratinocytes and fibroblasts (Misteli, 2007). Another possibility explaining differential regulation of gene expression by BMPs in distinct cell types could be the differences in their high-order chromatin structure and localisation of the BMP target genes relatively to distinct intra-nuclear functional compartments, such as eu- and hetero-chromatin, which may affect the accessibility of the targets for Smad transcription complexes (Lanctot et al., 2007).

In summary, we report here the first comprehensive comparative microarray analysis of global changes in gene expression programs induced by BMP-4 in human epidermal keratinocytes and dermal fibroblasts. We demonstrate that BMP-Smad pathway is active in both cell types and its activation is accompanied by distinct changes in transcriptomes of keratinocytes and fibroblasts, which are far more complex versus the effects of BMPs on cell proliferation/differentiation described previously. Furthermore, our data suggest that potential modulation of cell adhesion, extracellular matrix remodelling, cellular motility, metabolism, signalling and transcription by BMP-4 in keratinocytes and fibroblasts is likely to be achieved by the distinct sets of molecules specific for each cell type. Finally, these data provide an important platform for further delineating mechanisms that underlie the distinct effects of the BMP pathway on different cell populations in the skin and will be helpful in further establishing molecular signalling networks regulating skin homeostasis in health and disease.

Materials and methods

Cell culture experiments

Normal human epidermal keratinocytes and dermal fibroblasts were isolated from neonatal foreskin, as described previously (Gilchrest, 1979; Schindler et al., 2007). Foreskin tissue was obtained using a protocol approved by the University of Boston Institutional Review Board in accordance with HIPAA and the Declaration of Helsinki Principles. Keratinocytes were plated in serum-free keratinocytes medium (Defined Keratinocytes-SFM, Invitrogen, San Diego, CA) containing 0.15 mM calcium chloride and antibiotic-antimicotic mixture. Two days before the experiment the cells were put in the medium containing 0.05 mM calcium chloride. Neonatal fibroblasts were grown in Dulbecco’s Modification of Eagle’s Medium (DMEM, Invitrogen, San Diego, CA), containing 10% fetal bovine serum (HyClone, Logan, UT) and antibiotic-antimicotic mixture (100X; Invitrogen, San Diego, CA). Keratinocytes and fibroblasts were used in all the experiments at the third passage at 40–60% confluence. The cells were treated with 50 ng/ml recombinant human BMP-4 (R@D Systems, Minneapolis, MN) or diluent control. The fresh medium was added to the cells 4 hours before the BMP-4 addition.

Western blot analysis

Western blot analysis of total cellular proteins was performed, as described before (Sharov et al., 2005; Sharov et al., 2003). All experiments were performed using at least three to four replicates. Antibody reaction was performed with rabbit monoclonal anti-phospho-p38 MAPK (Cell Signalling, Danvers, MA), rabbit monoclonal anti-phospho ERK1/2 (Cell Signalling, Danvers, MA), rabbit polyclonal anti-p38 MAPK (Sigma, St Louise, MO), rabbit polyclonal anti-ERK1/2 (Sigma, St Louise, MO), rabbit polyclonal anti-phospho JNK (Sigma, St Louise, MO), rabbit polyclonal anti-JNK (,Sigma, St Louise, MO), mouse monoclonal anti-β-actin antibodies (Sigma, St Louise, MO) or rabbit polyclonal anti-phospho- SMAD1/5/8 antibodies (Millipore, Billerica, MA).

Microarray and RT-PCR analysis

Total RNA was isolated from neonatal keratinocytes and fibroblasts using TriIzol® Reagent (Invitrogen, San Diego, CA). All experiments were performed using at least three to four replicates, and RNA isolated from three-four experimental and control samples was pooled and processed for microarray analyses using one sample of pooled RNA per experimental and control group. All microarray analyses were performed at the Microarray Core Facility at Boston University School of Medicine using Human Genome U133 2.0 array (manufactured by Affymetrix) and the resulting data have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE16111 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16111). After statistical analysis and initial filtering the microarray data the changes in gene expression after BMP-4 treatment equal or higher 1.8 fold with at least one signal equal or higher than 80 fluorescence units for the gene was considered significant. For semi-quantitative and quantitative RT-PCR analysis equal amounts of total RNA was used as a template for cDNA synthesis using SupperScript III First-Strand Synthesis System and random primers (Invitrogene, San Diego, CA). PCR primers were designed using Beacon Designer software (Premier Biosoft International, Paolo Alto, CA) and are listed in Suppl. Table 4. Real time PCR was performed using iCycler Thermal Cycler (Bio-Rad Corp., Hercules, CA). Differences between samples and controls were calculated using Gene Expression Macro program (Bio-Rad Corp., Hercules, CA) based on the ΔΔCt equitation method.

Supplementary Material

Table S1
Table S2
Table S3
Table S4

Abbreviations

Act

Activin

BMP

Bone Morphogenetic Protein

FB

fibroblast

HF

Hair follicle

KC

keratinocyte

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1
NIHMS302246-supplement-Table_S1.doc (79.5KB, doc)
Table S2
NIHMS302246-supplement-Table_S2.doc (399.5KB, doc)
Table S3
NIHMS302246-supplement-Table_S3.doc (587.5KB, doc)
Table S4
NIHMS302246-supplement-Table_S4.doc (63KB, doc)

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