type viii collagen modulates tgf- 1-induced proliferation ...€¦ · collagen, a nonfibrillar...

Type VIII Collagen Modulates TGF-�1-inducedProliferation of Mesangial Cells

Ivonne Loeffler,* Ulrike Hopfer,† Dirk Koczan,‡ and Gunter Wolf*

*Department of Internal Medicine III, Friedrich Schiller University, Jena, Germany; †Department of Biomedicine,University of Basel, Basel, Switzerland; and ‡Institute for Immunology-Steinbeis Transfercenter for ProteomeAnalysis, University of Rostock, Rostock, Germany

ABSTRACTMesangial cells in diabetic mice and human kidneys with diabetic nephropathy exhibit increased type VIIIcollagen, a nonfibrillar protein that exists as a heterodimer composed of �1(VIII) and �2(VIII), encodedby Col8a1 and Col8a2, respectively. Because TGF-�1 promotes the development of diabetic glomeru-losclerosis, we studied whether type VIII collagen modulates the effects of TGF-�1 in mesangial cells. Weobtained primary cultures of mesangial cells from wild-type, doubly heterozygous (Col8a1�/�/Col8a2�/�),and double-knockout (Col8a1�/�/Col8a2�/�) mice. TGF-�1 bound normally to double-knockout mesan-gial cells. In wild-type mesangial cells, TGF-�1 inhibited proliferation, but in double-knockout cells, itstimulated proliferation, promoted cell cycle progression, and reduced apoptosis; we could reverse thiseffect by reconstituting �1(VIII). Furthermore, in wild-type cells, TGF-�1 mainly stimulated the Smadpathways, whereas in double-knockout cells, it activated the MAPK and PI3K/Akt pathways and inducedexpression of fibroblast growth factor 21 (FGF21). Inhibiting FGF21 expression by either interfering withactivation of the MAPK and PI3K/Akt pathways or by FGF21 siRNA attenuated the TGF-�1-inducedproliferation of double-knockout mesangial cells. In vivo, diabetic double-knockout mice had significantlyhigher expression of renal FGF21 mRNA and protein compared with diabetic wild-type mice. Immuno-histochemistry revealed strong expression of FGF21 in both glomerular (mesangial) and tubular cells ofdiabetic mice. Taken together, these data suggest that type VIII collagen significantly modulates theeffect of TGF-�1 on mesangial cells and may therefore play a role in the pathogenesis of diabeticnephropathy.

J Am Soc Nephrol 22: 649–663, 2011. doi: 10.1681/ASN.2010010098

Diabetic nephropathy is clinically characterized byproteinuria and progressive renal insufficiency andis the leading cause of end-stage renal failure in de-veloped countries.1 Podocyte injury, glomerularbasement membrane thickening, mesangial hyper-trophy, and expansion with subsequent develop-ment of glomerulosclerosis and tubulointerstitialfibrosis have long been recognized as pathologichallmarks.2,3 Several hormones, cytokines, andgrowth factors are known to play an important rolein the genesis and progression of diabetic nephrop-athy. In particular, the multifunctional cytokineTGF-�1 has been identified as stimulating the syn-thesis and inhibiting the degradation of extracellu-lar matrix molecules, and promoting mesangial cell

hypertrophy, one of the cardinal characteristics ofdiabetic glomerulopathy.4,5 It has been found thatTGF-�1 mRNA and protein expressions are ele-vated in the glomeruli and renal cortex of humanswith diabetic nephropathy and in various experi-mental models of diabetic nephropathy.6,7 On the

Received January 22, 2010. Accepted October 20, 2010.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Gunter Wolf, Department of InternalMedicine III, University of Jena, Erlanger Allee 101, D-07740Jena, Germany. Phone: 49-3641-9324301; Fax: 49-3641-9324302- E-mail: [email protected]

Copyright © 2011 by the American Society of Nephrology

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J Am Soc Nephrol 22: 649–663, 2011 ISSN : 1046-6673/2204-649 649

other hand, neutralization of TGF-�1 by an anti-TGF-� anti-body prevented glomerular hypertrophy and attenuated en-hanced extracellular matrix gene expression in experimentaldiabetic nephropathy.8,9 TGF-�1 initially stimulates mesangialcell proliferation but subsequently inhibits cell cycle progres-sion by induction of cell cycle inhibitors such as p27Kip1, re-sulting in mesangial hypertrophy.10 –12 Moreover, TGF-�1simulates the expression of key extracellular matrix moleculesincluding different types of collagen, fibronectin, and laminin,and suppresses, in parallel, the degradation of these mole-cules.5,13 This leads to the complex accumulation of matrix,resulting in diffuse glomerular sclerosis and progressive tubu-lointerstitial fibrosis.14

In addition to the classical collagen types I, III, IV, and VIfound in diabetic nephropathy, a strong specific induction of typeVIII collagen has been found in mesangial cells in diabetic miceand in kidney biopsies from patients with human diabetic ne-phropathy compared with normal kidneys.15,16 Type VIII colla-gen, a nonfibrillar collagen protein, exists as a heterodimer com-posed of two distinct polypeptide chains designated as �1(VIII)and �2(VIII) (genes: Col8a1 and Col8a2).17,18 Although increas-ing evidence indicates that type VIII collagen, expressed by a lim-ited number of cell types, may play an important role in mesangialcell function and the pathogenesis of diabetic glomerulosclerosis,the detailed physiologic function of this matrix protein remainsunclear. This study investigates the potential role of type VIII col-

lagen in modulating the biologic effects of TGF-�1 on mesangialcells.

RESULTS

TGF-�1 Induces the Expression of Col8a1 in MouseMesangial CellsPrevious studies have shown that high ambient glucose induces ex-pression of Col8a1 in mouse mesangial cells.15 Therefore, the effect ofrecombinant TGF-�1 was tested. Figure 1 shows that stimulationwith 2.5 ng/ml TGF-�1 for 24 hours significantly enhanced the ex-pression of Col8a1 mRNA in wild-type (by 78.2 � 7.8%; Figure 1A)as well as in Col8a1�/�/Col8a2�/� heterozygous (HZ) (by 77.5 �16.3%; Figure 1B) mesangial cells. No specific Col8a1 mRNA or�1(VIII) protein was detected in the absence or presence of TGF-�1in Col8a1�/�/Col8a2�/� knockout (KO) mesangial cells (Figure1, C and D). However, �1(VII) expression could be reconsti-tuted in KO mesangial cells using the mammalian expressionvector pCEP in which the Col8a gene transcription is driven bya cytomegalovirus promoter (Figure 1, C and D).15

Mesangial Cell Proliferation Is Induced by TGF-�1 inCol8a1�/�/Col8a2�/� KO Mesangial CellsWe have previously shown that TGF-�1 inhibits proliferationof mesangial cells and induces cell hypertrophy.10,11 Therefore,

Figure 1. Induction of Col8a1 mRNA expression in mesangial cells from wildtype (A) and Col8a1�/�/Col8a2�/� heterozygous mice (B).Values were normalized to 18S rRNA expression, and the control was assigned as an arbitrary value of 100% (n � 9). ***P � 0.001 versuscontrols). (C) No Col8a1 mRNA expression was found after RT-PCR in the absence or presence of TGF-�1 in Col8a1�/�/Col8a2�/� KOmesangial cells. However, Col8a1 mRNA expression can be reconstituted by the Col8a1/pCEP expression vector, whereas the emptypCEP vector was without effect. (D) Western blot for �1(VIII) collagen. No expression was found in Col8a1�/�/Col8a2�/� KO mesangialcells, but �1(VIII) collagen expression was reconstituted with the Col8a1/pCEP expression vector. The data are representative of twoseparate experiments.


650 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 649–663, 2011

we further investigated whether endogenous type VIII collagenexpression may modulate the influence of TGF-�1 on the pro-liferation. Wild-type, Col8a1�/�/Col8a2�/� HZ, and Col8a1�/�/Col8a2�/� KO mesangial cells were stimulated with 2.5 ng/mlTGF-�1 for 24 hours. Cell proliferation was assessed by directcell counting as well as DNA synthesis rate. Under these con-ditions, TGF-�1 significantly increased the total cell number ofCol8a1�/�/Col8a2�/� KO (by 37.5 � 7.3%), whereas no in-crease was seen in Col8a1�/�/Col8a2�/� HZ and wild-typemesangial cells (Figure 2A). To confirm these findings with analternative approach, we investigated the DNA synthesis rateunder similar conditions. Cells were labeled with bromode-oxyuridine (BrdU), and the incorporation into DNA was as-sessed. Induction of DNA synthesis by TGF-�1 was apparentin Col8a1�/�/Col8a2�/� KO, whereas the cytokine inhibitedDNA synthesis in Col8a1�/�/Col8a2�/� HZ as well as wild-type mesangial cells (Figure 2B). Forced reconstitution ofCol8a1 expression using a cytomegalovirus-promoter plas-mid, but not the empty control vector, in Col8a1�/�/Col8a2�/� KO cells abrogated the TGF-�1-induced prolif-eration (Figure 2C).

Col8a1 Expression Influences the Effect of TGF-�1 onCell Cycle and ApoptosisIt is well known that in many cell types, TGF-�1 can affect thecell cycle and induce apoptosis.11,19 To address this, we mea-sured cell cycle and apoptosis by fluorescence-activated cellsorter (FACS) analyses in mesangial cells exposed to 2.5 ng/mlTGF-�1 (Figure 3, A through G). Cell cycle analysis revealedthat TGF-�1 increased the number of Col8a1�/�/Col8a2�/�

KO cells in the S phase (by 81.4 � 17.0%), whereas in wild-typeas well as in Col8a1�/�/Col8a2�/� HZ mesangial cells, theDNA synthesis was significantly inhibited by TGF-�1 (Figure3, A through C and F). Moreover, TGF-�1 raised the numberof Col8a1�/�/Col8a2�/� KO cells in the G2/M phase (by38.5 � 7.2%) compared with wild-type cells (Figure 3, Athrough C and G), indicating completion of the cell cycle. Incontrast, TGF-�1-mediated cell cycle arrest in wild-type cellswas attenuated in Col8a1�/�/Col8a2�/� KO mesangial cells asdemonstrated by a reduction of the number of cells in the G1phase (by 18.9 � 5.0%) as well as in the sub-G1 phase (by24.6 � 9.8%) by treatment of TGF-�1 (Figure 3, A through E).Because cells in the sub-G1 phase may be apoptotic, we as-sessed annexin V-fluorescein binding and propidium-iodidestaining with FACS analysis (Figure 3H). TGF-�1 significantlyreduced the rate of apoptotic (annexin-positive but propidiumiodide-negative) Col8a1�/�/Col8a2�/� KO mesangial cellsrate (by 26 � 6%) in contrast to wild-type mesangial cells(Figure 3H).

The Receptor Affinity for TGF-�1 Is Not DifferentTo exclude the possibility that the different effects of TGF-�1may be caused by changes in the expression of receptors forTGF-�1, saturation binding studies with 125I-TGF-�1 wereperformed. Binding occurred in a concentration-dependent

Figure 2. TGF-�1-mediated effects on proliferation are mod-ified by the presence of �1(VIII) collagen. (A) Total cell numberis significantly stimulated by 2.5 ng/ml TGF-�1 for 24 hoursonly in Col8a1�/�/Col8a2�/� KO mesangial cells but not inwild-type or Col8a1�/�/Col8a2�/� HZ cells (n � 10 indepen-dent experiments with triplicates, ***P � 0.001 versus con-trols). (B) DNA synthesis determined as bromodeoxyuridineincorporation. Whereas 2.5 ng/ml TGF-�1 induced a significantdecrease in DNA synthesis in wild-type and Col8a1�/�/Col8a2�/� HZ cells, it clearly stimulated DNA synthesis inCol8a1�/�/Col8a2�/� KO mesangial cells (n � 10 independentexperiments done four times each, **P � 0.01 versus controlsof the same genotype). (C) Effect of a Col8a1 reconstitution oncell number. Col8a1�/�/Col8a2�/� KO mesangial cells weretransfected either with control plasmid pCEP or Col8a1/pCEP(each 0.8 and 1.6 �g/ml) for 12 hours and stimulated with 2.5ng/ml TGF-�1 for a further 24 hours. Reconstitution of Col8a1expression reduced the TGF-�1 induced proliferation to basallevels, whereas the empty control vector had no effect (n � 9,***P � 0.001 versus controls without TGF-�1, #P � 0.001versus cells transfected with control vector [1.6 �g/ml] treatedwith TGF-�1).

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manner over the range of 0.5 to 25 pM andwas saturable (data not shown). Scatchardanalysis of binding data revealed a singleclass of high affinity receptors for TGF-�1with similar dissociation constants (Kd)(Kd values of wild-type cells: 15.5 � 1.1 pM;Kd of Col8a1�/�/Col8a2�/� KO 14.2 � 0.9pM, n � 6, NS). Furthermore, maximalbinding capacity for TGF-�1 was equal inboth cell types (data not shown).

TGF-�1 Signaling Pathways DifferDepending on Type VIII CollagenExpressionBecause TGF-�1 can activate diverse sig-naling pathways including Smad and mito-gen-activated protein (MAP) kinase path-ways,20,21 we tested whether these secondmessengers are differentially regulated.Wild-type, Col8a1�/�/Col8a2�/� HZ, andCol8a1�/�/Col8a2�/� KO mesangial cellswere exposed to 2.5 ng/ml TGF-�1 for dif-ferent time periods, and cell extracts wereanalyzed by Western blotting with antibod-ies against Smad3, Erk 1,2, and Akt (Figure 4).In contrast to wild-type cells, TGF-�1 acti-vated in Col8a1�/�/Col8a2�/� KO cells ac-tivated MAP kinase and Akt pathwaysrather than Smad3 (Figure 4). Further-more, phosphorylated and total Erk 1,2 andPI3K/Akt was increased in Col8a1�/�/Col8a2�/� KO mesangial cells comparedwith the wild-type even in the absence ofTGF-�1.

Difference in TGF-�1-induced GeneExpression in Wild-type and Col8a1�/�/Col8a2�/� KO Mesangial CellsWe next assessed potential differences inthe TGF-�1-induced transcriptome inwild-type and Col8a1�/�/Col8a2�/� KOmesangial cells using microarray analysis.Out of the more than 39,000 transcripts onthe mouse Affymetrix array, 945 showed areliable signal. The number of genes in-duced or repressed at least two-fold was110. Gene array analysis was performed ac-cording the MIAME Guidelines,22,23 anddata have been submitted the microarry in-formatic database of the European Bioin-formatics Institute (accession numberE-MEXP-2658). Further information howthe analysis of gene arrays was performedand selected results are shown in the sup-plemental materials. Thirty-three known

Figure 3. The effect of TGF-b1 on cell cycle and apoptosis depends on the presence of typeVIII collagen. (A through C) Representative FL-2A histograms of Col8a1�/�/Col8a2�/� wild-type, Col8a1�/�/Col8a2�/� HZ, and Col8a1�/�/Col8a2�/� KO mesangial cells treated with2.5 ng/ml TGF-�1 or control medium. (D) Sub-G1 phase. TGF-�1 significantly reduced thenumber of Col8a1�/�/Col8a2�/� KO mesangial cells (n � 12, *P � 0.05 versus controls). (E) G1phase of cell cycle. Treatment of Col8a1�/�/Col8a2�/� KO mesangial cells with TGF-�1significantly reduced the number of cells in the G1 phase (n � 12, **P � 0.01 versus controls).(F) S phase. Whereas TGF-�1 significantly lowered the number of wild-type and Col8a1�/�/Col8a2�/� HZ mesangial cells in the S1 phase, it increased the number of Col8a1�/�/Col8a2�/� KO in the S phase (n � 12, *P � 0.05 versus controls, **P � 0.01 versus controls,***P � 0.001 versus controls of the same genotype). (G) TGF-�1 for 24 hours significantlyincreased only the number of Col8a1�/�/Col8a2�/� KO mesangial cells in the G2/M phase ofthe cell cycle (n � 12, ***P � 0.001 versus controls). (H) Apoptosis as determined by annexinV-FITC and propidium-iodide staining followed by FACS analysis. TGF-�1 significantly reducesapoptosis of Col8a1�/�/Col8a2�/� KO mesangial cells (n � 12, *P � 0.01 versus controls).



genes were upregulated, and 77 were down-regulated (data notshown). We found that genes involved in, for example, extra-cellular matrix formation, cell cycle, apoptosis, survival, MAPkinase signaling, and DNA replication showed altered expres-sion levels. Several genes (collagen type V [2.9-fold], laminin[3.4-fold], TGF-�1 [7.3-fold], TGF-� receptor type II [3.5-fold], fibroblast growth factor 2 [3.0-fold], fibroblast growthfactor (FGF21) [10.7-fold], and inhibitor of apoptosis protein[2.1-fold]) were highly induced by TGF-�1 in Col8a1�/�/Col8a2�/� KO compared with TGF-�1-challenged wild-typemesangial cells (Figure 5A). Real-time PCR analyses ofNF-�B and PI3K, both well known as important regulatorsof cell survival,24 confirmed the finding that TGF-�1 is ableto induce components of cell survival pathways in the ab-sence of type VIII collagen expression (increase of p110�expression by 40.9 � 12.7%) (Figure 5B).

TGF-�1-induced FGF21 Expression Is Increased in theAbsence of Type VIII CollagenAffymetrix analysis indicated that the TGF-�1-mediated FGF21gene expression is much higher in Col8a1�/�/Col8a2�/� KOcompared with TGF-�1-treated wild-type mesangial cells. Be-cause it has been recently reported that FGF21 is a novel metabolicfactor regulating glucose uptake,25 we further focused on this cy-tokine. As shown in Figure 6A, FGF21 mRNA expression wasinducible by TGF-�1 in all three mesangial cells, but induction ofFGF21 mRNA was significantly higher (approximately three-fold) in Col8a1�/�/Col8a2�/� KO mesangial cells than in wild-type cells. This finding was confirmed by Western blot analysis(Figure 6B). Time-course analysis of FGF21 expression inCol8a1�/�/Col8a2�/� KO mesangial cells showed that TGF-�1stimulated expression of FGF21 protein in a time-dependentmanner (Figure 6C). Next, we investigated whether the TGF-�1-

Figure 4. TGF-b1 induced signal transduction is different in wildtype and Col8a1�/�/Col8a2�/� KO mesangial cells. (A) Signaltransduction pathways. Wild-type, Col8a1�/�/Col8a2�/� HZ cells, and Col8a1�/�/Col8a2�/� KO mesangial cells were stimulated for5 minutes or 3 hours with 2.5 ng/ml with (�) or without (�) TGF-�1, and phosphorylation of Smad3, Erk1,2, and Akt was analyzed byWestern blots using phospho-specific antibodies. (B through D) Statistical analysis after densitometry of the blots. Whereas TGF-�1leads to phosphorylation of Smad3 and only relatively little phosphorylation of Erk1,2 and Akt in wild-type cells, MAP kinase activationand Akt phosphorylation was significantly stronger in Col8a1�/�/Col8a2�/� KO mesangial cells after challenge with TGF-�1 (C and D,*P �0.05 versus no TGF-�1, **P �0.01 versus no TGF-�1, n � 3 independent experiments). In contrast Smad3 phosphorylation wasattenuated. Col8a1�/�/Col8a2�/� cells compared with the wild-type (B, *P �0.05 versus no TGF-�1, **P �0.01 versus no TGF-�1, n �3 independent experiments) HZ mesangial cells revealed an intermediate response. The blots are representative for three independentexperiments with qualitatively similar results.



induced FGF21 expression is dependent on Erk 1,2 and PI3K/Aktpathways. Interference with the Erk 1,2 (with PD98059) as well asPI3K/Akt (with LY294002) pathways attenuated the TGF-�1-mediated upregulation of FGF21 mRNA Col8a1�/�/Col8a2�/�

KO mesangial cells (Figure 6D). Moreover, TGF-�1-inducedFGF21 protein was reversed to basal levels by PD 098059 orLY294002 (Figure 6E). This Western blot also clearly demon-strates that the inhibitors prevented phosphorylation of Erk 1,2and Akt (Figure 6E). To test the cell specificity of the TGF-�1-induced FGF21 mRNA expression, podocytes as well as a mousemesangial cell line (MMC) were studied.26 As shown in Figure 6F,TGF-�1 significantly stimulated FGF21 mRNA expression inmesangial cells with the highest induction in Col8a1�/�/Col8a2�/� KO mesangial cells but not in podocytes.

Inhibition of FGF21 Expression Attenuates theTGF-�1-mediated Proliferation in Col8a1�/�/Col8a2�/�

KO Mesangial CellsInhibition of Erk 1,2 or PI3K/Akt pathways attenuated theTGF-�1-stimulated proliferation of Col8a1�/�/Col8a2�/� KOmesangial cells (Figure 7A). To further investigate the role ofFGF21 in the TGF-�1-induced proliferation of Col8a1�/�/Col8a2�/� KO mesangial cells, FGF21 expression was inhib-ited by siRNA. 5 and 10 nM FGF21 siRNA, but not controlsiRNA, inhibited TGF-�1-mediated FGF21 protein expressionin Col8a1�/�/Col8a2�/� KO mesangial cells (Figure 7B). Inhi-bition of FGF21 protein expression using specific siRNA inhib-ited the TGF-�1-stimulated proliferation of Col8a1�/�/Col8a2�/� KO mesangial cells (Figure 7C).

Enhanced Renal FGF21 Expression in DiabeticCol8a1�/�/Col8a2�/� KO MiceTo address the in vivo situation, we investigated the levels ofFGF21 in serum and kidney homogenate in the streptozoto-

cin-induced diabetic wild-type, Col8a1�/�/Col8a2�/� HZ,and Col8a1�/�/Col8a2�/� KO mice. Because it has been re-cently demonstrated that serum FGF21 levels and FGF21mRNA expression in different tissues are increased in obeseand diabetic mice,27 we used db/db diabetic mice as a control.Serum FGF21 concentrations significantly increased only indb/db mice, a model of type2 diabetes, compared with nondi-abetic db/m littermates, whereas no significant difference wasobserved in streptozotocin-induced diabetic mice comparedwith nondiabetic controls (Figure 8A). However, renal FGF21mRNA (Figure 8B) and protein (Figure 8C) expression wassignificantly higher in diabetic Col8a1�/�/Col8a2�/� KO micecompared with diabetic wild-type animals.

DISCUSSION

Mesangial cells play an important role in the development of dia-betic glomerulosclerosis. In vitro and in vivo studies have demon-strated that mesangial cells exposed to high glucose or the diabeticenvironment initially undergo a very limited proliferation andbecome subsequently arrested in the G1 phase of the cell cycle.10,26

This G1 phase arrest is associated with the development of cellularhypertrophy and production of extracellular matrix proteinsleading to mesangial expansion and sclerosis.28 TGF-�1 is a keyfactor induced by high glucose, mediating G1 phase arrest by in-duction of cell cycle inhibitors such as p27Kip1.11,12

We have recently found in biopsies from patients with dia-betic nephropathy that type VIII collagen mRNA and proteinexpression (Fig. 9) is upregulated compared with healthy con-trol kidneys.16 Furthermore, type VIII collagen expression inhuman diabetic nephropathy was not correlated with renalfunction and proteinuria, suggesting that the diabetic environ-ment itself may be primarily responsible for the induction of

Figure 5. Differential expression of target genes in wildtype and Col8a1�/�/Col8a2�/� KO mesangial cells after stimulation withTGF-�1. (A) Affymetrix analysis of eight selected genes from 33 induced by 2.5 ng/ml TGF-�1 for 24 hours in Col8a1�/�/Col8a2�/� KOcompared with wild-type mesangial cells (n � 3). Col5a1, �1(V) collagen; Lama, laminin; TGFBRII, TGF-beta receptor type II; FGF2,fibroblast growth factor 2. (B) Quantitative RT-real-time PCR for p110�, a catalytic subunit of PI3K. TGF-�1 significantly stimulatedp110� mRNA expression only in Col8a1�/�/Col8a2�/� KO mesangial cells (n � 12, ***P � 0.001 versus controls of the same genotype).



expression.16 In fact, high glucose-containing medium in-duced Col8a1 mRNA expression independent of its osmolarityin a MMC, but not in murine tubular and glomerular endo-thelial cells.15 Col8a2 was not induced by high glucose in mes-angial cells. Further studies in STZ-induced diabetic Col8a1�/�/Col8a2�/� KO mice revealed less mesangial matrix depositionand albuminuria compared with diabetic wild-type animals.Because it has been previously shown that type VIII collagenmay stimulate migration of smooth vascular muscle cells29 butmay inhibit proliferation of endothelial cells,30 this study ad-dresses whether type VIII collagen modulates the influence ofTGF-�1, one of the major cytokines in diabetic nephropathy,on cell cycle regulation in mesangial cells. We used mesangialcells isolated from genetically modified mice deficient in bothCol8 genes to test whether there is a relationship between thebiologic effects of exogenous TGF-�1 and the expression oftype VIII collagen.31 We provide evidence that TGF-�1 in-duced proliferation of mesangial cells in the absence of endog-enous type VIII collagen expression. TGF-�1-induced signaltransduction changed from utilization of the Smad system toactivation of Erk 1,2 and PI3K/Akt pathways in the absence oftype VIII collagen, but TGF-�1-receptor affinity and expres-sion was not modulated. Limited GeneChip analysis revealedthat TGF-�1-induced FGF21 expression is markedly increasedin mesangial cells deficient of type VIII collagen. A greaterrenal expression of FGF21 was also seen in diabetic Col8a1�/�/Col8a2�/� KO mice compared with diabetic wild-type ani-mals. FGF21 is a recently discovered novel endocrine and para-crine metabolic regulator.32 When given to diabetic animals,FGF21 stimulates glucose uptake into cells and lowers plasmaglucose as well as triglycerides.33 On the other hand, FGF21mRNA expression itself is induced in the liver by glucose.34

Stimulation of the PI3K/Akt signaling pathway inducesFGF21.35 However, Wente et al. have previously demonstratedthat FGF21 stimulates insulin gene transcription and insulinbiosynthesis via activation of Erk 1,2 signaling in the pancreatic�-cell, promotes �-cell survival, and inhibits �-cell apoptosisvia activation of PI3K/Akt signaling, which resulted in a strongreduction in circulating glucose levels and an increased num-ber of islets and �-cells in diabetic mice after long-term admin-istration of FGF21.36 Renal effects of FGF21 have not beenpreviously described. FGF21 signals through cell-surface re-ceptors composed of the classic FGF receptors complexed with�-Klotho but can also act through all four FGR isotypes.37–39

Klotho is highly expressed in the kidney and mesangial cells,and these cells have been found to also express FGF recep-tors.40,41 Thus, our findings that FGF21 mediates the TGF-�1induced proliferation in Col8a1�/�/Col8a2�/� KO mesangialcells are in accordance with these earlier findings. Because ithas been previously shown that FGF21 expression is inducedby the PI3K/Akt pathway and there are multiple interactionsbetween the PI3K/Akt and Erk 1,2 pathways,35,42,43 the shiftfrom TGF-�1-induced Smad activation to the noncanonicalErk 1,2 and PI3K/Akt in the absence of type VIII collagen is thecause of mesangial proliferation. However, the increase in

Figure 6. (A through E) FGF21 expression induced by TGF-beta isstrongest in Col8a1�/�/Col8a2�/� KO mesangial cells. (A) AlthoughTGF-�1 for 24 hours increased FGF21 mRNA expression in all threecell types, the induction was much stronger in Col8a1�/�/Col8a2�/�

KO mesangial cells. Quantitative RT-real-time PCR normalized to 18S rRNA (n � 12, *P � 0.01 versus controls, **P � 0.001 versuscontrols of the same phenotype). (B) Western blot analysis for FGF21protein expression. Stimulation of wild-type mesangial cells for 24hours with TGF-�1 resulted in no detectable band, whereasCol8a1�/�/Col8a2�/� HZ and Col8a1�/�/Col8a2�/� KO mesangialcells clearly expressed FGF21 protein after TGF-�1 treatment. Thisblot is representative of three independent experiments with quali-tatively similar results. (C) Time-course analysis of FGF21 proteinexpression in Col8a1�/�/Col8a2�/� KO mesangial cells. The cellswere stimulated with 2.5 ng/ml TGF-�1 (�) or control medium with-out TGF-�1 (�) for the indicated length of time, and FGF21 proteinexpression was analyzed by Western blot. TGF-�1-mediated FGF21protein expression occurred after 3 hours, peaked at 6 to 9 hours, anddiminished after 24 hours. This blot is representative of two separateexperiments.



whole kidney FGF21mRNA expression as well the enhancedserum concentration of FGF21 in diabetic animal is likely dueto stimulated tubular expression as detected in the immuno-histochemistry. Nevertheless, the discrete increase in mesan-gial cell staining for FGF21 in diabetic Col8a1�/�/Col8a2�/�

KO mice but not in the diabetic wild-type is presumably suffi-cient to stimulate local mesangial proliferation.

How exactly type VIII collagen modulates TGF-�1-associ-ated signal transduction pathways is currently unclear, but afew suggestions can be made. Older studies have shown that

Figure 6. (continued) (D) Effect of MAP kinase (PD98059, PD) or PI3K inhibitors (LY294002, LY) on TGF-�1-induced FGF21 mRNAexpression Col8a1�/�/Col8a2�/� KO mesangial cells. The cells were preincubated with 20 �M PD 98059 and 10 �M LY294002 for 30minutes and then stimulated with TGF-�1 for further 24 hours. Both inhibitors significantly reduced the TGF-�1-mediated increase inFGF21 mRNA (n � 8, *P � 0.001 versus controls, #P � 0.01 versus TGF-�1 only). (E) Representative blots of FGF21 protein expressionin the presence or absence of MAP kinase (PD98059) or PI3K inhibitors (LY294002) in Col8a1�/�/Col8a2�/� KO mesangial cells. Thecells were treated with TGF-�1 (2.5 ng/ml) for the indicated times. Either 20 �M PD98059 or 10 �M LY294002 were given for 30 minutesbefore TGF-�1 treatment. PD98059 inhibits TGF-�1 phosphorylation of Erk 1,2 and LY294002 Akt phosphorylation, respectively.TGF-�1 induced FGF21 protein expression was abolished by PD98059 as well as LY294002. This blot is representative of threeindependent experiments. (F) Quantification of ERK phosphorylation in Col8a1�/�/Col8a2�/� KO mesangial cells (**P � 0.01 versustime-matched controls without TGF-b1, ***P � 0.001 versus time-matched controls without TGF-b1, n�3). (G) Quantification of AKTphosphorylation in Col8a1�/�/Col8a2�/� KO mesangial cells (**P � 0.01 versus time-matched controls without TGF-b1, ***P � 0.001versus time-matched controls without TGF-b1, n � 3). (H) FGF21 mRNA expression is induced by TGF-b1 in mesangial cells. Primarymesangial cells (wildtype and Col8a1�/�/Col8a2�/� KO), a mouse mesangial cell line (MMC), and mouse podocytes were stimulatedwith TGF-b1 for 24 hours and the FGF21 mRNA expression was determined with RT-real-time PCR analysis. Data of relative mRNAexpression are normalized to 18S rRNA. TGF-b1 induced only in mesangial cells FGF21 mRNA with the strongest increase inCol8a1�/�/Col8a2�/� KO cells (n � 12, **P � 0.01 versus controls, ***P � 0.001 versus controls of the same genotype, #P �0.01 versusCol8a1�/�/Col8a2�/� wildtype mesangial cells treated with TGF-b1).



collagen type V is required for the growth and inhibitory ac-tions of TGF-�1 nontransformed epithelial cells.44 The NC1domain of the �1 chain of type VIII collagen inhibits the pro-liferation of bovine aortic endothelial cells, but the mecha-nisms behind these anti-proliferative effects were not furtherinvestigated.30 Several studies have also demonstrated that var-ious collagens can exert anti-proliferative effects on mesangialcells.45,46 For example, collagen type I suppresses Erk 1,2 acti-vation and is antiproliferative for mesangial cells.46 Discoidindomain receptors (DDR1 and 2) are receptor tyrosine kinasesthat function as receptors for various collagens.47 DDR1 isexpressed on mesangial cells, and type VIII collagen binds toand activates this receptor.48,49 Interestingly, Curat and Vo-gel report that DDR1-knockout mesangial cells exhibited anincreased proliferation and activation of Erk 1,2.50 Thus, thelack of type VIII collagen synthesis after TGF-�1 stimula-tion in Col8a1�/�/Col8a2�/� KO mesangial cells may leadto a reduced DDR1 activation with concomitant stimula-

tion of the Erk 1,2 pathway. This, together with the inducedFGF21 synthesis and activation of other pathways (e.g. NF-�B), ultimately leads to TGF-�1-induced proliferation andnot hypertrophy in Col8a1�/�/Col8a2�/� KO mesangialcells. Further studies that are beyond the scope of this reportare necessary to define the exact role of DDR1 in TGF-�1-induced proliferation of Col8a1�/�/Col8a2�/� KO mesan-gial cells.

Is TGF-�1-mediated mesangial proliferation in the ab-sence of type VIII collagen better than hypertrophy? Regres-sion of diabetic mesangial matrix expansion and glomeru-losclerosis have occurred in humans and in experimentalmodels of diabetic nephropathy.51,52 It is reasonable to as-sume that this process requires some limited proliferationof mesangial cells to restore an intact environment besidesproteolytic clearance of the deposited extracellular matrixproteins. Diabetic p27Kip1 knockout mice have a reducedglomerular matrix expansion and less albuminuria despite

Figure 7. TGF-�1-induced proliferation depends on FGF21 expression. (A) Inhibition of MAP kinases with PD98059 or PI3K withLY294002 completely attenuates TGF-�1-inuduced proliferation of Col8a1�/�/Col8a2�/� KO mesangial cells (n � 24, ***P �0.001 versus control, #P � 0.001 versus TGF-�1 only). (B) FGF21 siRNA inhibits TGF-�1-induced FGF21 protein expression inCol8a1�/�/Col8a2�/� KO mesangial cells. A control siRNA was without effect. The blot is representative of two independentexperiments. (C) Transfection of Col8a1�/�/Col8a2�/� KO mesangial cells with FGF21 siRNA, but not with control siRNA, inhibitsTGF-�1-induced proliferation (n � 12, ***P � 0.001 versus controls; #P � 0.01 versus TGF-�1 only, ##P � 0.001 versus TGF-�1only). (D) TGF-�1-induced a modest proliferative effect in heterozygous Col8a1�/�/Col8a2�/� (*P � 0.05 versus no TGF-�1, n �12) and a stronger increase in cell number in Col8a1�/�/Col8a2�/� KO mesangial cells (**P � 0.01 versus no TGF-�1, n � 12).These effects were antagonized by FGF21 siRNA (##P � 0.01 versus control siRNA, n � 12). Neither the control siRNA nor theFGF21 siRNA exhibited any influence on cell number of Col8a1�/�/Col8a2�/� wild-type mesangial cells in the presence orabsence of TGF-�1 (n � 12).



an increase in the mesangial cell number compared withdiabetic wild-type animals.53 Bone marrow-derived cellscan differentiate into mesangial cells after renal injury.54

Bone-marrow infusion can ameliorate progressive glomer-ulosclerosis in different experimental models.55,56 Althoughde novo formation of endothelial cells is important in thisprocess, there is experimental evidence that the repair alsoinvolves mesangial cells.57 Glomerular mesangial cells pro-vide structural support, and a repair process after diabetic

nephropathy likely requires a well-balanced recruitment ofglomerular endothelial and mesangial cells. Alternatively,one may see the increase in type VIII collagen expressionduring diabetic nephropathy as an intrinsic renal attempt tolimit progression of disease.

In summary, we found that TGF-�1 induces a prolifera-tion of mesangial cells in the absence of concomitant typeVIII collagen expression. This effect is in strong contrast towild-type mesangial cells in which TGF-�1 is anti-prolifer-

Figure 8. FGF21 expression in diabetic mice is significantly higher in diabetic mice. (A) Serum FGF21 concentrations were onlysignificantly enhanced in diabetic db/db mice compared with nondiabetic db/� animals. STZ-induced diabetes in wild-type, Col8a1�/�/Col8a2�/� HZ, and Col8a1�/�/Col8a2�/� KO mice did not significantly change serum FGF21 concentrations (n � 5 animals in each group,*P � 0.01 versus db/�). (B) Renal FGF21 mRNA expression. FGF21 transcripts were highest in STZ-induced diabetic Col8a1�/�/Col8a2�/�

KO and db/db mice (n � 5 animals per group, *P � 0.05 versus nondiabetic controls of the same genotype, **P � 0.01 versus nondiabeticcontrols of the same genotype). (C) Western blot analysis of FGF21 protein expression in whole renal homogenates from diabetic mice. Ingeneral expression of FGF21 protein was significantly higher in diabetic mice (STZ or db/db) compared with nondiabetic controls (*P � 0.05versus nondiabetic controls, n � 2). In addition, bands for FGF21 protein are significantly stronger in homogenates from diabeticCol8a1�/�/Col8a2�/� KO mice compared with diabetic wild-type mice (#P � 0.05 versus diabetic wild-type, n � 2).



ative and induces cell cycle arrest. The proliferative effect ofTGF-�1 in Col8a1�/�/Col8a2�/� KO mesangial cells is as-sociated with a shift from Smad to noncanonical TGF-�1-pathways and depends on the induction of FGF21.

CONCISE METHODS

Animal ExperimentsAll of the animal experiments were approved by the Thuringer Lande-

samt fur Lebensmittelsicherheit und Verbraucherschutz and done in ac-

cordance with the German Animal Protection Law. The generation and

phenotype of Col8a1�/�/Col8a2�/� KO mice has been previously de-

scribed in detail.31 Col8a1�/�/Col8a2�/� KO and Col8a1�/�/Col8a2�/�

HZ mice were crossed back for at least 20 generations into the C57Bl/6

background,31 and wild-type mice were maintained in a pathogen-free

facility. All of the animals had free access to water and were on the

standard rodent chow. Ten- to twelve-week-old mice were randomly

divided into groups treated with streptozotocin (STZ; Sigma, St.

Louis, MO) or left untreated. STZ was dissolved in sterile 10 mM

sodium citrate (pH 5.5) and injected intraperitoneally (50 mg/kg

body wt) for five consecutive days. To render the animals hypergly-

cemic without becoming ketoacidotic, a subcu-

taneous insulin implant (LinShin, Toronto,

Canada) was administered.15 Venous blood glu-

cose concentrations were measured with Free

Style Lite (Abbott Diabetes Care, Wiesbaden,

Germany). The mice were sacrificed after 40

days. In addition, diabetic db/db mice and con-

trol db/m mice (Jackson Laboratory, Bar Har-

bor, ME) were used at the age of 4 months. At

the end of the experiment, venous blood was

collected in tubes on ice containing aprotinin

(0.6 TIU/ml of blood; Phoenix Pharmaceuticals,

Burlingame, CA) to inhibit the activity of pro-

teinases. FGF21 protein concentrations were

determined in serum samples using a mouse

FGF21 RIA kit (Phoenix Pharmaceuticals, Bur-

lingame, CA). In addition, the kidneys were har-

vested and mechanically homogenized with the

homogenizer SpeedMill P12 (Analytik Jena Bio

Solutions, Jena, Germany), and the total RNA

and protein were isolated as described below.

Isolation and Culture of MesangialCellsMesangial cells were obtained from the isolated

glomeruli of Col8a1�/�/Col8a2�/� wild-type,

Col8a1�/�/Col8a2�/� HZ, and Col8a1�/�/

Col8a2�/� KO mice kidneys according to previ-

ously described protocols of sieving and centrif-

ugation for the preparation of primary

mesangial cell cultures.15,25 Our studies used

subcultures of the fifteenth to twentieth pas-

sages of mesangial cells grown in RPMI 1640

medium (Promo Cell, Heidelberg, Germany),

containing 5 mM glucose and 10% heat-inactivated FCS at 37°C in

5% CO2. Routine identification of the mesangial cells was performed

by indirect immunofluorescence microscopy using antibodies against

�-smooth-muscle actin (Progen Biotechnik, Heidelberg, Germany),

synaptopodin (Sigma), E-cadherin (Abcam, Cambridge, UK), and

PECAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA). Used cells

stained positive for �-smooth-muscle actin but were negative for syn-

aptopodin, E-cadherin, and PECAM-1. The cells were seeded in ap-

propriate tissue culture plates and cultured for 24 hours. The cells

were serum-starved (0.1% FCS) overnight and exposed as described

for the indicated lengths of time.

Proliferation AssaysA colorimetric immunoassay kit for BrdU incorporation (Roche Di-

agnostics, Mannheim, Germany) was used for quantification of DNA

synthesis.58 1 � 103 cells were seeded in 96-well plates; after incuba-

tion in RPMI supplemented with 0.1% FCS for 24 hours, the cells

were treated with 2.5 ng/ml human recombinant TGF-�1 (Peprotech,

Hamburg, Germany) for 24 hours. Mesangial cells were then labeled

with BrdU for 4 hours at 37°C, and the incorporation was determined

with the assay kit following the manufacturer’s instructions. The ab-

Figure 9. Increase in FGF21 mesangial expression only in diabetic Col8a1�/�/Col8a2�/�

KO mice. (A through H) Immunohistology for FGF21-protein expression. (A) Negativecontrol with normal rabbit serum. (B) Nondiabetic Col8a1�/�/Col8a2�/� wild-type mice.(C) Diabetic Col8a1�/�/Col8a2�/� wild-type animals. (D) Nondiabetic Col8a1�/�/Col8a2�/� heterozygous mice. (E) Diabetic Col8a1�/�/Col8a2�/� heterozygous mice. (F)Nondiabetic Col8a1�/�/Col8a2�/� KO mice. (G) Diabetic Col8a1�/�/Col8a2�/� KO mice.(H) Nondiabetic db/� mice. (I) diabetic db/db mice (at 4 weeks). Although all diabeticanimals revealed an increase in tubular staining for FGF21, a weak increase in mesangialFGF21 expression was only found in diabetic Col8a1�/�/Col8a2�/� KO mice (G). Inter-estingly, diabetic db/db mice showed an increase in FGF21 expression in visceral cells ofBowman’s capsule. No specific staining was found with nonimmune serum (A). Thisstaining is representative of five sections obtained from each kidney from five individualanimals with qualitatively similar results (magnification, �400).



sorbance of the samples was measured at 450 nm using a microplate

reader (Tecan, Crailsheim, Germany).

For automated cell counting, 5 � 104 cells were seeded in 12-well

culture plates and cultured for 24 hours. The cells were serum-starved

overnight and exposed to agonists for a further 24 hours. The cells

were detached from the culture dishes by trypsinization and counted

in a CASY Cell Counter (Scharfe System, Reutlingen, Germany) using

the Analyzer System Model DT routine according to the manufacturer’s

instructions.

For reconstitution experiments, full-length Col8a1 cDNA15

cloned into pCEP-Pu vector or pCEP-Pu control vector were trans-

fected into mesangial cells (80 to 90% confluence) using Lipofecta-

minTM 2000 reagent (Invitrogen, Karlsruhe, Germany) according to

the manufacturer’s protocol.

Cell Cycle Analysis and ApoptosisCell cycle analysis and apoptosis assay were performed using flow cytom-

etry. After 24 hours of treatment with 2.5 ng/ml TGF-�1, the cells were

harvested by trypsinization and washed twice with PBS. The cells were

suspended in 200 �l of PBS with 2% FCS and incubated in staining

solution (10 mM 1,4-piperazinediethanesulfonic acid, 2 mM MgCl2, 0.1

M NaCl, 0.1% Triton X-100, 25 �g/ml RNase [Roche Diagnostics], 10

�g/ml propidium iodide [Sigma]) at 4°C for 30 minutes in the dark.

Then each sample was analyzed using a FACScalibur flow cytometer

(Becton Dickinson, Franklin Lake, NJ), and the percentages of cells

within the sub-G1, G1, S, and G2/M phases of the cell cycle were deter-

mined. The total number of cells analyzed for each sample was 10,000,

and raw data were processed using CellQuestPro and WinMDI software.

Annexin V binding was determined with an apoptosis assay kit

(Roche Diagnostics) following the manufacturer’s instructions. Flu-

orescence was measured on the FACScalibur flow cytometer.

TGF-�1 Receptor Binding Assay5 � 104 Col8a1�/�/Col8a2�/� wild-type and Col8a1�/�/Col8a2�/�

KO mesangial cells were plated per well in a 12-well plate and grown

for 24 hours. After serum deprivation for 12 hours, the cells were

washed once with ice-cold PBS and preincubated with binding buffer

(serum-free RPMI with 5 mM MgCl2, 10 mM 6-aminocaproic acid, 1

mM phenylmethylsulfonyl fluoride, 0.05% aprotinin, and 1 mg/ml

bovine serum albumin) on a shaking platform at 4°C for 30 minutes.

For saturation binding assays, increasing amounts (0.5 to 25 pM) of

human recombinant 125I-TGF-�1 (specific activity, 3287 Ci/mmol)

(PerkinElmer, Waltham, MA) were added in binding buffer. Nonspe-

cific binding was determined in the presence of 0.5 to 25 nM human

recombinant TGF-�1 (Peprotech, Hamburg, Germany). After incu-

bation on a shaking platform at 4°C for 1 hour, the cells were gently

washed three times with ice-cold binding buffer and lysed with 1 ml of

5 M NaOH with 0.5% Triton X-100. The amount of radioactivity was

counted in a gamma scintillation counter. Control wells without ra-

dioactivity were trypsinized, and the cell number was determined

with a CASY Cell Counter (Scharfe System, Reutlingen, Germany).

Nonspecific binding was subtracted, and the data were analyzed with

GraphPad Prism (GraphPad Software, La Jolla, CA). The results are

presented as Scatchard plots of saturation binding data, and each

point represents the mean of three individual experiments.

RNA Extraction, cDNA Synthesis, and ReverseTranscription (RT) Real-time PCRFor RT real-time PCR analysis, total RNA was isolated from treated

cells or kidney homogenates using the RNeasy kit (Qiagen, Hilden,

Germany), and 1 �g was reverse-transcribed using the Reverse Tran-

scription System (Promega, Madison, WI) following the manufacturer’s

instructions. The expression levels of genes were determined by quan-

titative PCR using LightCycler-FastStart DNA Master SYBR Green I

(Roche Diagnostics) on a Real-Plex Mastercycler (Eppendorf, Ham-

burg, Germany). PCRs were carried out in a total volume of 20 �l

containing 2 �l of cDNA and sense and antisense primers in a con-

centration of 0.25 �M each. To normalize for differences in the

amount of cDNA in each sample, we performed amplification of 18 S

ribosomal RNA as an endogenous control. The amplification pro-

gram included an initial denaturation step (10 minutes, 95°C), 40

cycles of amplification (denaturation for 10 seconds at 95°C), primer

annealing (temperature see Table 1) for 15 seconds, extension for 10

seconds at 72°C, an additional heating step to melt potential primer

dimers for 5 seconds at 95°C), and a melting curve program (dena-

turation for 10 seconds at 95°C, cooling and holding for 15 seconds at

60°C, and then heating at a speed of 0.1°C/s to 95°C). Finally, a cooling

step of 2 minutes at 40°C was performed. Table 1 shows the sequences

of the primer pairs. The transcript levels were normalized to the mean

value of samples from the unstimulated controls.

Microarray ExperimentsAfter stimulation of Col8a1�/�/Col8a2�/� wild-type and Col8a1�/�/

Col8a2�/� KO mesangial cells with 2.5 ng/ml TGF-�1 for 24 hours,

total RNA was extracted as described above. The GeneChip Mouse

Genome 430 2.0 Array containing probes of over 39000 transcripts

(Affymetrix, Santa Clara, CA) was used for analysis. Array hybridiza-

tion was performed according to the supplier’s instructions using the

GeneChip Expression 3�Amplifikation One-Cycle Target Labeling

and Control reagents from Affymetrix. The scanning of the microar-

ray was done with the GeneChip Scanner 3000 (Affymetrix) at 1.56 �

resolution. Data analysis was performed with the MAS 5.0 (Microar-

Table 1. List of primer pairs and annealing temperature

Gene Sense Primers Antisense Primers Tann.

18 S rRNA 5�-CACGGCCGGTACAGTGAAAC-3� 5�-GATCTGATAAATGCACGC-3� 58°CCol8a1 5�-CGGGAGTAGGAAAACCAGGAGTG-3� 5�-CTCGGCCCAAGAACCCCAGGAA-3� 62°CFGF21 5�-ATGGAATGGATGAGATCTAGAGTTGG-3� 5�-TCTTGGTGGTCATCTGTGTAGAGG-3� 62°CNF�B 5�-AGTACCTGCCAGATACAGACGAT-3� 5�-GATGGTGCTCAGGGATGACGTA-3� 58°Cp110� 5�-GAACCAGTAGGCAACCGTGAA-3� 5�-GCATCCTCCCCAGCATTAT-3� 56°CVinculin 5�-GCCAAGCAGTGCACAGATAA-3� 5�-TTCCTTTCTGGTGTGTGAAGC-3� 62°C



ray Suite statistical algorithm; Affymetrix) probe level analysis using

GeneChip Operating Software (GCOS 1.4), and the final data extrac-

tion was done with the DataMining Tool 3.1 (Affymetrix). Each ex-

periment (stimulation of cells, isolation of RNA, chip hybridization,

and analysis) was independently performed three times, and only dif-

ferentially expressed transcripts in all sets of experiments were further

analyzed. For the details of data analysis according to the MIAME

Guidelines,22,23 see the online supplemental materials.

FGF21 siRNATo inhibit the FGF21 mRNA expression, mesangial cells were trans-

fected with siRNA targeting FGF21 (5�-TTGGAATAATAAA-

GAGTCTGA-3�; NM_020013) or control siRNA (Qiagen) using

HiPerfect Transfection Reagent (Qiagen). After incubation with

the transfection complexes under normal growth conditions over-

night, the medium was changed, and the cells were stimulated with

2.5 ng/ml TGF-�1 for 24 hours.

Western Blot AnalysisCultured mesangial cells and kidney homogenate were lysed using Com-

plete LysisM (Roche Diagnostics) and supplemented with the phospha-

tase inhibitor sodium orthovanadate (100 �M). Lysates were cleared by

centrifugation, and 50 �g of protein/lane was separated by SDS-PAGE.

Western blotting was performed as described previously.59 The primary

antibodies used were as follows: anti-mouse collagen �1(VIII) (Abcam,

Cambridge, UK), anti-phospho-Erk 1,2, anti-phospho-Akt, anti-

phospho-Smad3 (Cell Signaling Technology, Danvers, MA), and anti-

mouse FGF21 (R&D Systems, Minneapolis, MN). The blots were subse-

quently reprobed with anti-�-vinculin (Sigma), anti-Erk 2, anti-Akt

(Santa Cruz Biotechnology), and anti-Smad3 (Cell Signaling Technol-

ogy). Peroxidase-labeled secondary antibodies to rabbit IgG and mouse

IgG (1:10,000) were purchased from KPL (Gaithersburg, MD). Western

Lightning Chemiluminescence Reagent Plus (ECL; PerkinElmer LAS,

Boston, MA) was used for detection of signals, and the images were cap-

tured using a Fuji LAS-3000 imaging system (Fujifilm Life Science, Dus-

seldorf, Germany) as described previously.58,59

Immunohistochemistry for FGF21 ExpressionParaffin-embedded kidneys were sectioned at 4 �m. For the detection of

FGF21, a rabbit polyclonal to FGF21 (Abcam, Cambridge, UK) with a

concentration of 0.30 mg/ml was used in a 1.100 dilution (this antibody

reacts with human and murine FGF21). Detection was performed with

the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) using an

anti-rabbit secondary antibody as described previously.59

Statistical AnalysesThe values given in this article are presented as the means � SEM. The

results were analyzed with the Kruskal-Wallis test for multigroup

comparison followed by the Mann-Whitney U-test. A P value of

�0.05 was considered significant.

ACKNOWLEDGMENTS

We would like to acknowledge Simone Schonfelder and Sybille

Franke for excellent support, Martin Forster for help with FACS anal-

ysis, Claus Liebmann for helpful discussions of the receptor binding

studies, and Wolfgang Schmidt-Heck for the help with the data anal-

ysis of microarray studies. This work was supported by a grant from

the Deutsche Forschungsgemeinschaft to G.W.

DISCLOSURESNone.

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type viii collagen modulates tgf- 1-induced proliferation ...€¦ · collagen, a nonfibrillar...

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