Skin Fibrosis and Recovery Is Dependent on Wnt Activation via DPP4

Fibrosis is the life-threatening, excessive accumulation of the extracellular matrix and is sometimes associated with a loss of lipid-filled cells in the skin and other organs. Understanding the mechanisms of fibrosis and associated lipodystrophy and their reversal may reveal new targets for therapeutic intervention. In vivo genetic models are needed to identify key targets that induce recovery from established fibrosis. Wnt signaling is activated in animal and human fibrotic diseases across organs. Here, we developed a genetically inducible and reversible Wnt activation model and showed that it is sufficient to cause fibrotic dermal remodeling, including extracellular matrix expansion and shrinking of dermal adipocytes. Upon withdrawal from Wnt activation, Wnt-induced fibrotic remodeling was reversed in mouse skin—fully restoring skin architecture. Next, we demonstrated CD26/ DPP4 is a Wnt/β-catenin–responsive gene and a functional mediator of fibrotic transformation. We provide genetic evidence that the Wnt/DPP4 axis is required to drive fibrotic dermal remodeling and is associated with human skin fibrosis severity. Remarkably, DPP4 inhibitors can be repurposed to accelerate recovery from established Wnt-induced fibrosis. Collectively, this study identifies Wnt/DPP4 axis as a key driver of extracellular matrix homeostasis and dermal fat loss, providing therapeutic avenues to manipulate the onset and reversal of tissue fibrosis. Fibrosis is the life-threatening, excessive accumulation of the extracellular matrix and is sometimes associated with a loss of lipid-filled cells in the skin and other organs. Understanding the mechanisms of fibrosis and associated lipodystrophy and their reversal may reveal new targets for therapeutic intervention. In vivo genetic models are needed to identify key targets that induce recovery from established fibrosis. Wnt signaling is activated in animal and human fibrotic diseases across organs. Here, we developed a genetically inducible and reversible Wnt activation model and showed that it is sufficient to cause fibrotic dermal remodeling, including extracellular matrix expansion and shrinking of dermal adipocytes. Upon withdrawal from Wnt activation, Wnt-induced fibrotic remodeling was reversed in mouse skin—fully restoring skin architecture. Next, we demonstrated CD26/ DPP4 is a Wnt/β-catenin–responsive gene and a functional mediator of fibrotic transformation. We provide genetic evidence that the Wnt/DPP4 axis is required to drive fibrotic dermal remodeling and is associated with human skin fibrosis severity. Remarkably, DPP4 inhibitors can be repurposed to accelerate recovery from established Wnt-induced fibrosis. Collectively, this study identifies Wnt/DPP4 axis as a key driver of extracellular matrix homeostasis and dermal fat loss, providing therapeutic avenues to manipulate the onset and reversal of tissue fibrosis. Excessive deposition of extracellular matrix (ECM) proteins leads to scarring and fibrosis, inducing tissue stiffening and loss of function in virtually all organ systems, including the skin, adipose tissue, heart, intestine, and lungs (Distler et al., 2019Distler J.H.W. Györfi A.H. Ramanujam M. Whitfield M.L. Königshoff M. Lafyatis R. Shared and distinct mechanisms of fibrosis.Nat Rev Rheumatol. 2019; 15: 705-730Crossref PubMed Scopus (224) Google Scholar). Despite its devastating impact on nearly 5% of people worldwide annually (Zhao et al., 2020Zhao X. Kwan J.Y.Y. Yip K. Liu P.P. Liu F.F. Targeting metabolic dysregulation for fibrosis therapy.Nat Rev Drug Discov. 2020; 19: 57-75Crossref PubMed Scopus (161) Google Scholar), no effective treatment for fibrosis exists. Interestingly, fibrosis occurs concomitantly with a loss of lipid-filled cells in several organs, including adipocytes in the adipose tissue and skin, and lipo-fibroblasts in the lungs and liver (DeBari and Abbott, 2020DeBari M.K. Abbott R.D. Adipose tissue fibrosis: mechanisms, models, and importance.Int J Mol Sci. 2020; 21: E6030Crossref PubMed Scopus (38) Google Scholar; El Agha et al., 2017El Agha E. Moiseenko A. Kheirollahi V. De Langhe S. Crnkovic S. Kwapiszewska G. et al.Two-way conversion between lipogenic and myogenic fibroblastic phenotypes marks the progression and resolution of lung fibrosis [published correction appears in Cell Stem Cell 2017;20:571.Cell Stem Cell. 2017; 20 (261–73.e3)Google Scholar; Hernandez-Gea and Friedman, 2011Hernandez-Gea V. Friedman S.L. Pathogenesis of liver fibrosis.Annu Rev Pathol. 2011; 6: 425-456Crossref PubMed Scopus (1264) Google Scholar; Rehan and Torday, 2014Rehan V.K. Torday J.S. The lung alveolar lipofibroblast: an evolutionary strategy against neonatal hyperoxic lung injury.Antioxid Redox Signal. 2014; 21: 1893-1904Crossref PubMed Scopus (38) Google Scholar; Schmidt and Horsley, 2013Schmidt B.A. Horsley V. Intradermal adipocytes mediate fibroblast recruitment during skin wound healing.Development. 2013; 140: 1517-1527Crossref PubMed Scopus (197) Google Scholar). The skin is an excellent system to study fibrosis because it is easily accessible and has distinct ECM and dermal white adipose tissue (DWAT) layers. Fibroblasts and adipocytes arise from common progenitor populations in the skin, enabling both cell types to be targeted when designing genetic models of skin fibrosis (Atit et al., 2006Atit R. Sgaier S.K. Mohamed O.A. Taketo M.M. Dufort D. Joyner A.L. et al.Beta-catenin activation is necessary and sufficient to specify the dorsal dermal fate in the mouse.Dev Biol. 2006; 296: 164-176Crossref PubMed Scopus (280) Google Scholar; Jiang et al., 2018Jiang D. Correa-Gallegos D. Christ S. Stefanska A. Liu J. Ramesh P. et al.Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring.Nat Cell Biol. 2018; 20: 422-431Crossref PubMed Scopus (65) Google Scholar; Rinkevich et al., 2015Rinkevich Y. Walmsley G.G. Hu M.S. Maan Z.N. Newman A.M. Drukker M. et al.Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.Science. 2015; 348: aaa2151Crossref PubMed Scopus (333) Google Scholar; Shook et al., 2018Shook B.A. Wasko R.R. Rivera-Gonzalez G.C. Salazar-Gatzimas E. López-Giráldez F. Dash B.C. et al.Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.Science. 2018; 362: eaar2971Crossref PubMed Scopus (156) Google Scholar). Dermal fibroblasts regulate ECM homeostasis including proteoglycans, providing structural integrity to the skin (Hunzelmann et al., 1996Hunzelmann N. Anders S. Sollberg S. Schönherr E. Krieg T. Co-ordinate induction of collagen type I and biglycan expression in keloids.Br J Dermatol. 1996; 135: 394-399Crossref PubMed Scopus (45) Google Scholar). Dermal adipocytes are dynamic in the skin and are associated with the hair cycle, as they undergo hypertrophy during hair growth (Festa et al., 2011Festa E. Fretz J. Berry R. Schmidt B. Rodeheffer M. Horowitz M. et al.Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling.Cell. 2011; 146: 761-771Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar) and shrink in association with hair follicle regression (Festa et al., 2011Festa E. Fretz J. Berry R. Schmidt B. Rodeheffer M. Horowitz M. et al.Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling.Cell. 2011; 146: 761-771Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar; Nicu et al., 2019Nicu C. Hardman J.A. Pople J. Paus R. Do human dermal adipocytes switch from lipogenesis in anagen to lipophagy and lipolysis during catagen in the human hair cycle?.Exp Dermatol. 2019; 28: 432-435Crossref PubMed Scopus (22) Google Scholar; Zhang et al., 2019bZhang Z. Shao M. Hepler C. Zi Z. Zhao S. An Y.A. et al.Dermal adipose tissue has high plasticity and undergoes reversible dedifferentiation in mice.J Clin Invest. 2019; 129: 5327-5342Crossref PubMed Scopus (77) Google Scholar). In addition, adipocytes impact thermoregulation (Alexander et al., 2015Alexander C.M. Kasza I. Yen C.L. Reeder S.B. Hernando D. Gallo R.L. et al.Dermal white adipose tissue: a new component of the thermogenic response.J Lipid Res. 2015; 56: 2061-2069Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar; Kasza et al., 2014Kasza I. Suh Y. Wollny D. Clark R.J. Roopra A. Colman R.J. et al.Syndecan-1 is required to maintain intradermal fat and prevent cold stress.PLoS Genet. 2014; 10e1004514Crossref Scopus (73) Google Scholar) and can produce antimicrobial peptides in skin infection models and can also regulate inflammation after injury (Driskell et al., 2014Driskell R.R. Jahoda C.A. Chuong C.M. Watt F.M. Horsley V. Defining dermal adipose tissue.Exp Dermatol. 2014; 23: 629-631Crossref PubMed Scopus (183) Google Scholar; Shook et al., 2018Shook B.A. Wasko R.R. Rivera-Gonzalez G.C. Salazar-Gatzimas E. López-Giráldez F. Dash B.C. et al.Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.Science. 2018; 362: eaar2971Crossref PubMed Scopus (156) Google Scholar; Zhang et al., 2015Zhang L.J. Guerrero-Juarez C.F. Hata T. Bapat S.P. Ramos R. Plikus M.V. et al.Innate immunity. Dermal adipocytes protect against invasive Staphylococcus aureus skin infection.Science. 2015; 347: 67-71Crossref PubMed Scopus (290) Google Scholar). Both adipocytes and fibroblasts are essential to the mechanical properties and function of the skin (Butzelaar et al., 2017Butzelaar L. Niessen F.B. Talhout W. Schooneman D.P.M. Ulrich M.M. Beelen R.H.J. et al.Different properties of skin of different body sites: the root of keloid formation?.Wound Repair Regen. 2017; 25: 758-766Crossref PubMed Scopus (12) Google Scholar; Ezure and Amano, 2010Ezure T. Amano S. Influence of subcutaneous adipose tissue mass on dermal elasticity and sagging severity in lower cheek.Skin Res Technol. 2010; 16: 332-338Crossref PubMed Scopus (28) Google Scholar; Ezure and Amano, 2015Ezure T. Amano S. Increment of subcutaneous adipose tissue is associated with decrease of elastic fibres in the dermal layer.Exp Dermatol. 2015; 24: 924-929Crossref PubMed Scopus (45) Google Scholar; Wollina et al., 2017Wollina U. Wetzker R. Abdel-Naser M.B. Kruglikov I.L. Role of adipose tissue in facial aging.Clin Interv Aging. 2017; 12: 2069-2076Crossref PubMed Scopus (40) Google Scholar). Skin fibrosis, with the expansion of dermal ECM and reduction of DWAT, likely has profound impacts on these functions. To advance our understanding of tissue fibrosis, we need to identify signals and specific mediators that can affect multiple cell types in fibrosis, so that we can harness their therapeutic potential to reverse fibrotic remodeling. Several signaling pathways have been implicated in fibrosis development (Distler et al., 2019Distler J.H.W. Györfi A.H. Ramanujam M. Whitfield M.L. Königshoff M. Lafyatis R. Shared and distinct mechanisms of fibrosis.Nat Rev Rheumatol. 2019; 15: 705-730Crossref PubMed Scopus (224) Google Scholar; Piersma et al., 2015Piersma B. Bank R.A. Boersema M. Signaling in fibrosis: TGF-β, WNT, and YAP/TAZ converge.Front Med (Lausanne). 2015; 2: 59Crossref PubMed Scopus (305) Google Scholar), including TGFβ and PDGFRα signaling (Marcelin et al., 2017Marcelin G. Ferreira A. Liu Y. Atlan M. Aron-Wisnewsky J. Pelloux V. et al.A PDGFRα-mediated switch toward CD9high adipocyte progenitors controls obesity-induced adipose tissue fibrosis.Cell Metab. 2017; 25: 673-685Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar; Olson and Soriano, 2009Olson L.E. Soriano P. Increased PDGFRalpha activation disrupts connective tissue development and drives systemic fibrosis.Dev Cell. 2009; 16: 303-313Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Although less well studied, canonical Wnt signaling through its transducer, β-catenin, is a conserved stimulus of tissue fibrosis in many organs, including the skin (Akhmetshina et al., 2012Akhmetshina A. Palumbo K. Dees C. Bergmann C. Venalis P. Zerr P. et al.Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.Nat Commun. 2012; 3: 735Crossref PubMed Scopus (585) Google Scholar; Beyer et al., 2013Beyer C. Reichert H. Akan H. Mallano T. Schramm A. Dees C. et al.Blockade of canonical Wnt signalling ameliorates experimental dermal fibrosis.Ann Rheum Dis. 2013; 72: 1255-1258Crossref PubMed Scopus (99) Google Scholar; Hamburg-Shields et al., 2015Hamburg-Shields E. DiNuoscio G.J. Mullin N.K. Lafyatis R. Atit R.P. Sustained β-catenin activity in dermal fibroblasts promotes fibrosis by up-regulating expression of extracellular matrix protein-coding genes.J Pathol. 2015; 235: 686-697Crossref PubMed Google Scholar; Hu et al., 2020Hu H.H. Cao G. Wu X.Q. Vaziri N.D. Zhao Y.Y. Wnt signaling pathway in aging-related tissue fibrosis and therapies.Ageing Res Rev. 2020; 60: 101063Crossref PubMed Scopus (72) Google Scholar). While activation of Wnt signaling in skin fibroblasts has been shown in human fibrotic tissues, chemically induced fibrosis in mice, and constitutive activation of β-catenin in dermal fibroblasts in genetic mouse models can induce fibrosis (Akhmetshina et al., 2012Akhmetshina A. Palumbo K. Dees C. Bergmann C. Venalis P. Zerr P. et al.Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.Nat Commun. 2012; 3: 735Crossref PubMed Scopus (585) Google Scholar; Hamburg-Shields et al., 2015Hamburg-Shields E. DiNuoscio G.J. Mullin N.K. Lafyatis R. Atit R.P. Sustained β-catenin activity in dermal fibroblasts promotes fibrosis by up-regulating expression of extracellular matrix protein-coding genes.J Pathol. 2015; 235: 686-697Crossref PubMed Google Scholar; Hu et al., 2020Hu H.H. Cao G. Wu X.Q. Vaziri N.D. Zhao Y.Y. Wnt signaling pathway in aging-related tissue fibrosis and therapies.Ageing Res Rev. 2020; 60: 101063Crossref PubMed Scopus (72) Google Scholar; Wei et al., 2012Wei J. Fang F. Lam A.P. Sargent J.L. Hamburg E. Hinchcliff M.E. et al.Wnt/β-catenin signaling is hyperactivated in systemic sclerosis and induces Smad-dependent fibrotic responses in mesenchymal cells.Arthritis Rheum. 2012; 64: 2734-2745Crossref PubMed Scopus (184) Google Scholar), the cellular and molecular mechanisms by which Wnt signaling promotes fibrotic phenotypes are not well understood. Recent studies have demonstrated that En1+ embryonic mesenchymal progenitors can produce dermal fibroblasts and adipocytes and are responsible for ECM production in the skin’s dermis during development, homeostasis, and in response to fibrotic stimuli (Atit et al., 2006Atit R. Sgaier S.K. Mohamed O.A. Taketo M.M. Dufort D. Joyner A.L. et al.Beta-catenin activation is necessary and sufficient to specify the dorsal dermal fate in the mouse.Dev Biol. 2006; 296: 164-176Crossref PubMed Scopus (280) Google Scholar; Jiang et al., 2018Jiang D. Correa-Gallegos D. Christ S. Stefanska A. Liu J. Ramesh P. et al.Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring.Nat Cell Biol. 2018; 20: 422-431Crossref PubMed Scopus (65) Google Scholar; Mascharak et al., 2021Mascharak S. desJardins-Park H.E. Davitt M.F. Griffin M. Borrelli M.R. Moore A.L. et al.Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.Science. 2021; 372eaba2374Crossref PubMed Scopus (145) Google Scholar; Rinkevich et al., 2015Rinkevich Y. Walmsley G.G. Hu M.S. Maan Z.N. Newman A.M. Drukker M. et al.Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.Science. 2015; 348: aaa2151Crossref PubMed Scopus (333) Google Scholar; Shook et al., 2018Shook B.A. Wasko R.R. Rivera-Gonzalez G.C. Salazar-Gatzimas E. López-Giráldez F. Dash B.C. et al.Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.Science. 2018; 362: eaar2971Crossref PubMed Scopus (156) Google Scholar). To determine if activation of Wnt signaling in En1+ embryonic mesenchymal progenitors is sufficient to induce skin fibrosis and required to maintain fibrotic phenotypes, we generated an inducible and reversible genetic mouse model to express stabilized β-catenin in En1 lineage–derived mesenchymal cells. We found that several aspects of fibrotic remodeling, including dermal ECM remodeling and lipodystrophy, are induced and dependent on sustained Wnt signaling activation in mesenchymal cells. To identify mechanisms by which Wnt signaling activates fibrotic phenotypes, we screened for functional mediators with roles in adipocyte and fibroblast biology. We identified dipeptidyl peptidase 4 (DPP4) as a Wnt-responsive protein required for fibrotic remodeling of the dermis and dermal adipocyte lipodystrophy during fibrosis. We further showed that pharmacological inhibition of DPP4 accelerates recovery from fibrotic ECM and DWAT remodeling in mouse skin. Collectively, these data show that the Wnt-DPP4 axis is a key regulator of fibrotic dermal transformation and recovery from established Wnt-induced fibrosis. To determine whether Wnt activation in mesenchymal lineages is sufficient to induce and maintain fibrotic ECM and DWAT remodeling, we generated a mouse model with inducible and reversible β-catenin stabilization in the skin. To this end, we induced Wnt signaling activation in the previously characterized embryonic En1 lineage–derived mesenchymal cells, which produce all forms of dermal fibroblasts and adipocyte stem cells, and are profibrotic during skin injury and melanoma-induced fibrosis (Atit et al., 2006Atit R. Sgaier S.K. Mohamed O.A. Taketo M.M. Dufort D. Joyner A.L. et al.Beta-catenin activation is necessary and sufficient to specify the dorsal dermal fate in the mouse.Dev Biol. 2006; 296: 164-176Crossref PubMed Scopus (280) Google Scholar; Jiang et al., 2018Jiang D. Correa-Gallegos D. Christ S. Stefanska A. Liu J. Ramesh P. et al.Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring.Nat Cell Biol. 2018; 20: 422-431Crossref PubMed Scopus (65) Google Scholar; Mascharak et al., 2021Mascharak S. desJardins-Park H.E. Davitt M.F. Griffin M. Borrelli M.R. Moore A.L. et al.Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.Science. 2021; 372eaba2374Crossref PubMed Scopus (145) Google Scholar; Rinkevich et al., 2015Rinkevich Y. Walmsley G.G. Hu M.S. Maan Z.N. Newman A.M. Drukker M. et al.Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.Science. 2015; 348: aaa2151Crossref PubMed Scopus (333) Google Scholar; Shook et al., 2018Shook B.A. Wasko R.R. Rivera-Gonzalez G.C. Salazar-Gatzimas E. López-Giráldez F. Dash B.C. et al.Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.Science. 2018; 362: eaar2971Crossref PubMed Scopus (156) Google Scholar). We used the En1-Cre line to recombine the Rosa26 reverse tetracycline regulator transactivator (R26rtTA), including newly En1 positive and En1 lineage-derived cells. In the presence of dietary doxycycline, R26rtTA transactivates the TetO myc-tagged stabilized β-catenin (β-catistab), the signal transducer of activated canonical Wnt signaling (Figure 1a). We confirmed myc-tag and nuclear β-catenin expression in postnatal dermal fibroblasts and the dermal adipocyte layer in mouse skin (Supplementary Figure S1). β-catistab caused significant dermal ECM expansion concomitantly with a significant decrease in DWAT thickness within 10 days of doxycycline feeding (Figure 1b and c). Quantitative histomorphometry revealed early DWAT remodeling in fibrotic mouse skin, but we further investigated whether Wnt activation also induced changes in dermal adipocyte size. In early Wnt-induced fibrosis, mature adipocytes in β-catistab mice displayed reduced size of PLIN1+ lipid droplets, despite the increase in adipocyte lipid content that is associated with hair follicle growth (Festa et al., 2011Festa E. Fretz J. Berry R. Schmidt B. Rodeheffer M. Horowitz M. et al.Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling.Cell. 2011; 146: 761-771Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar) stimulated by sustained Wnt activation (Figure 1b and d). Hair follicle number is comparable between controls and β-catistab mouse dorsal skin (Supplementary Figure S4). These data indicated that mature adipocytes are dynamically decreasing in size early in response to Wnt activation. During the early stages of Wnt induction (10 days), we also found active collagen remodeling in the DWAT layer, though not in the rest of the dermis, as seen by significantly elevated collagen hybridizing peptide staining in the DWAT region (Figures 1d and 4c and Supplementary Figure S3). With sustained Wnt activation for 21 days, β-catistab mice had progressive dermal expansion and DWAT lipodystrophy, an increase in percent area of high-density collagen matrix and proteoglycans, and increased collagen fiber thickness (Figure 1b, c, and e and Supplementary Figure S4a and b). Elastin protein distribution is comparable in control and Wnt activated mice (Supplementary Figure S4d). Notably, DWAT remains diminished and shrunken adipocytes persist even after 21 days of Wnt activation (Figure 1b and d and Supplementary Figure S2). Because Wnt signaling activation in En1-lineage–derived mesenchymal cells led to progressive fibrotic remodeling, we next investigated which aspects of the fibrotic phenotype were dependent on Wnt signaling. To this end, we examined ECM and DWAT remodeling during reversal from β-catistab that is initiated by the removal of doxycycline (Figure 1b and c). β-catistab mice were fed doxycycline for 21 days and subsequently given normal chow for an additional 21 days (reversal) (Figure 1b and c). In the reversal phase, the expression of myc-tagged, stabilized β-catenin protein was absent within 10 days (Supplementary Figure S1). Reversal of Wnt activation in β-catistab mice remarkably restored DWAT layer thickness, dermal thickness, percent area high-density collagen matrix, proteoglycan area, and collagen fiber thickness to comparable levels to those measured in control mice (Figure 1b and c and Supplementary Figure S4). Thus, those aspects of fibrotic remodeling of the dermis and DWAT are dependent on sustained Wnt signaling activation. Taken together, these data indicate that fibrotic remodeling is dependent on sustained Wnt signaling activation in En1-lineage–derived mesenchymal cells. Although Wnt signaling has been implicated in fibrosis of various tissues, its functional effectors are unknown. To define the mechanisms by which Wnt/β-cat signaling promotes fibrotic remodeling, we analyzed transcriptional changes associated with induction of stabilized β-catenin from primary dermal fibroblasts (Adeno-Cre; β-cateninflox3/+) (GSE 103870) (Mullin et al., 2017Mullin N.K. Mallipeddi N.V. Hamburg-Shields E. Ibarra B. Khalil A.M. Atit R.P. Wnt/β-catenin signaling pathway regulates specific lncRNAs that impact dermal fibroblasts and skin fibrosis.Front Genet. 2017; 8: 183Crossref PubMed Scopus (15) Google Scholar). One of the 20 most differentially expressed genes that were highly upregulated (×47, P < 0.05) in an unbiased screen in β-catflox3/+ dermal fibroblasts was Dpp4, a multifunctional integral membrane glycoprotein and secreted serine protease which is expressed by many cell types (Gorrell, 2005Gorrell M.D. Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders.Clin Sci (Lond). 2005; 108: 277-292Crossref PubMed Scopus (287) Google Scholar; Röhrborn et al., 2016Röhrborn D. Brückner J. Sell H. Eckel J. Reduced DPP4 activity improves insulin signaling in primary human adipocytes.Biochem Biophys Res Commun. 2016; 471: 348-354Crossref PubMed Scopus (32) Google Scholar) (Figure 2a and b ). In other, nonfibrotic contexts, DPP4 has been shown to both promote lipid accumulation and cause adipocyte dedifferentiation in vitro (Lessard et al., 2015Lessard J. Pelletier M. Biertho L. Biron S. Marceau S. Hould F.S. et al.Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.PLoS One. 2015; 10e0122065Crossref Scopus (40) Google Scholar; Rosmaninho-Salgado et al., 2012Rosmaninho-Salgado J. Marques A.P. Estrada M. Santana M. Cortez V. Grouzmann E. et al.Dipeptidyl-peptidase-IV by cleaving neuropeptide Y induces lipid accumulation and PPAR-γ expression.Peptides. 2012; 37: 49-54Crossref PubMed Scopus (37) Google Scholar). DPP4 was of particular interest because it is also expressed in mouse fibrotic fibroblasts, where it has been linked to fibrotic ECM expansion (Mascharak et al., 2021Mascharak S. desJardins-Park H.E. Davitt M.F. Griffin M. Borrelli M.R. Moore A.L. et al.Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.Science. 2021; 372eaba2374Crossref PubMed Scopus (145) Google Scholar; Rinkevich et al., 2015Rinkevich Y. Walmsley G.G. Hu M.S. Maan Z.N. Newman A.M. Drukker M. et al.Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.Science. 2015; 348: aaa2151Crossref PubMed Scopus (333) Google Scholar; Shook et al., 2018Shook B.A. Wasko R.R. Rivera-Gonzalez G.C. Salazar-Gatzimas E. López-Giráldez F. Dash B.C. et al.Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.Science. 2018; 362: eaar2971Crossref PubMed Scopus (156) Google Scholar). Though DPP4 has been shown to play a role in skin repair and chemical models of fibrosis, previous studies neither identified the upstream regulators of its increased expression nor examined its role in fibrosis-associated lipodystrophy (Mascharak et al., 2021Mascharak S. desJardins-Park H.E. Davitt M.F. Griffin M. Borrelli M.R. Moore A.L. et al.Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.Science. 2021; 372eaba2374Crossref PubMed Scopus (145) Google Scholar; Rinkevich et al., 2015Rinkevich Y. Walmsley G.G. Hu M.S. Maan Z.N. Newman A.M. Drukker M. et al.Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.Science. 2015; 348: aaa2151Crossref PubMed Scopus (333) Google Scholar). First, we validated that DPP4 mRNA was Wnt responsive in β-catistab dermal fibroblasts in vitro and in mouse skin (Figure 2b–d). DPP4 protein expression was elevated within 5 days of β-catistab, before dermal remodeling, and remained elevated at 21 days of β-catistab in mouse skin (Figure 2c and Supplementary Figure S5). Elevated DPP4 immunoreactivity was present throughout the dermis, including in the DWAT layer (Figure 2c and Supplementary Figure S5). In addition, it was increased in bleomycin subcutaneous-injected skin within 5 days (Supplementary Figure S5). Wnt signaling activation with a Wnt agonist CHIR99021 also induced Dpp4 mRNA expression in mature dermal adipocytes in vitro in a cell-autonomous manner (Supplementary Figure S7). Together, these data show that DPP4 expression is Wnt-responsive in dermal mesenchymal cells. We also found that DPP4 immunoreactivity is elevated in human systemic sclerosis (SSc), corresponding to elevated dermal β-catenin (Figure 3a and b ). Interestingly, DPP4 immunoreactivity corresponds with SSc disease severity compared with control human skin (Figure 3b). Analysis of human DPP4 promoter sequence (within 5 KB) reveals predicted TCF/LEF family transcription factor binding sites (Figure 3c). This indicates that DPP4 is a relevant target in mouse fibrosis models, as well as in severe human SSc. Next, we tested the hypothesis that DPP4 is required to mediate Wnt-induced fibrotic remodeling of both dermal ECM and DWAT. To test this hypothesis, we examined whether genetic deletion of DPP4 rescues Wnt-induced fibrotic remodeling. Despite comparable Wnt activation, indicated by nuclear β-catenin and myc tag protein expression, in both Dpp4+/+ and Dpp4–/–; β-catistab mice, Dpp4–/–; β-catistab mice displayed increased PLIN1+ lipid vesicle size, and ultimately DWAT preservation (Figure 4a–c and Supplementary Figure S6a). This was accompanied by significantly reduced collagen remodeling in the DWAT and diminished collagen remodeling throughout the dermis in Dpp4–/–; β-catistab mice in early Wnt-induced fibrosis, resulting in attenuated dermal thickening, and a comparable proportion of high-density collagen matrix and similar collagen fiber thickness to controls (Figure 4b and c and Supplementary Figure S6). These results indicate that deletion of DPP4 protects against Wnt-induced fibrosis and particularly against fibrosis-associated lipodystrophy of DWAT. DPP4 functions as a soluble and cell surface protein with enzymatic and nonenzymatic functions (Gorrell, 2005Gorrell M.D. Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders.Clin Sci (Lond). 2005; 108: 277-292Crossref PubMed Scopus (287) Google Scholar; Röhrborn et al., 2016Röhrborn D. Brückner J. Sell H. Eckel J. Reduced DPP4 activity improves insulin signaling in primary human adipocytes.Biochem Biophys Res Commun. 2016; 471: 348-354Crossref PubMed Scopus (32) Google Scholar). To determine if inhibition of DPP4 enzymatic activity accelerates recovery from Wnt signaling activation induced fibrosis, we blocked DPP4’s enzymatic activity with the pharmacological inhibitor, sitagliptin (DPP4i) in vitro and in vivo (Dobrian et al., 2011Dobrian A.D. Ma Q. Lindsay J.W. Leone K.A. Ma K. Coben J. et al.Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice.Am J Physiol Endocrinol Metab. 2011; 300: E410-E421Crossref PubMed Scop