Transforming growth factor-β1 acts cooperatively with sodium n-butyrate to induce differentiation of normal human keratinocytes

EXPERIMENTAL

CELL RESEARCH

198,27-30

(1992)

Transforming Growth Factor-P1 Acts Cooperatively with Sodium nButyrate to Induce Differentiation of Normal Human Keratinocytes GENG WANG,* PAUL J. HIGGINS,? MAUREEN GANNON,* AND LISA STAIANO-COICO*~’ *Department of Surgery and Field of Cell Biology and Genetics of tke Graduate School of Medical Sciences, Cornell University Medical College, 1300 York Avenue, New York, New York 10021 and; iDepartment of Microbiology and Immunology, Albany Medical College, Albany, New York 12208

relatively low levels of spontaneous cornified envelopes (CEs) (~10%). Epidermal differentiation, however, can be positively influenced by sodium butyrate (NaB) which both induces progression to the CE stage and “desensitizes” NHEKs to the inhibitory influence of retinoids on epidermal maturation [ 1,2]. After a 4-day exposure to NaB, CE formation is dramatically increased (>50%) and is preceded by: (1) a block in the Gl and G2/M phases of the cell cycle; (2) increases in transglutaminase activity and involucrin expression; and, (3) cell type-specific cytoarchitectural reorganization [ 1,2]. CE induction by NaB is strictly associated with an up-regulation of transforming growth factor-p (TGF-P) mRNA specifically within the maturing suprabasal compartment, suggesting that TGF-j3 may play a role in the differentiation response of NHEKs to NaB 111. While TGF-j3 is known to reversibly inhibit cell cycle progression of NHEKs, its role in NHEK differentiation remains unclear [3-61. In uitro, it appears to exert pleiotropic effects on various aspects of the differentiation pathway [5-71. In normal epidermis in viuo, TGFpl is apparent only in the nonproliferating suprabasal NHEK, analogous to its location in NaB-treated NHEKs [8, 91. The present investigation was designed to elucidate the role of exogenous and endogenous TGF+l on terminal differentiation of NHEKs in response to NaB. We provide evidence that TGF-#I1 is necessary, but not sufficient, for the induction of mature CEs in NHEKs using the NaB differentiation model system.

Growth factors with established biological activity toward cultured normal human epidermal keratinocytes (NIIEKs) (e.g., transforming growth factor-& TGF-B; retinoic acid, RA) initiate programmed changes in cellular maturation which differ with regard to the specific differentiation pathway (normal or abnormal) analyzed. Sodium butyrate (NaR) initiates one form of epidermal differentiation leading to enhanced cornified envelope (CE) formation which involves abrogation of the normally inhibitory e5ect of RA on NIIEK maturation. NaR also induces TGF-/3 mRNA in the maturing suprabasal compartment, suggesting that TGF-B may play a role in NaR-initiated NHEK differentiation. Treatment with TGF+l alone, however, only marginally increased (by twofold) the number of detergent-resistant CEs compared to control NHEKs and did not alter the prevalence of fully mature enucleated CEs. TGF-@l was quite effective in inducing significant levels of CE expression when used simultaneously with suboptimal concentrations of NaR. The cooperative action of suboptimal NaR and TGF-Bl generated numbers of CEs which, in fact, exceeded the incidence of mature CEs formed in response to optimal levels of NaR alone. Neutralizing antibodies to TGF-8, moreover, e5ectively reduced the incidence of CE formation in cultures treated with optimal NaR concentrations, further implicating endogenous TGF-fl activity in the NaR-initiated NIIEK di5erentiation model. It is suggested, therefore, that within the NaILinduced pathway of NIIEK difPerentiation, TGF-fl can positively modulate expression of the differentiated phenotype but alone is insufficient for generation of mature CEs. o 1992 Academic

MATERIALS

Praw. Inc.

Keratinocyte culture. NHEKs were derived from cadaver skin and processed as previously described [lo]. After culture for a minimum of 7-10 days, TGF-j% (l-2 rig/ml; R-D Systems, CA) was added either alone or in combination with NaB (0.5-1.0 m&f); l-3 rig/ml TGF+l produced a maximal response which was not further enhanced by addition of higher concentrations of TGF-@l. NaB concentrations were chosen based upon dose-response curves. Maximal CE induction is observed with 3.0 m&f NaB [l]. Suboptimal doses (0.51.0 n&) of NaB were chosen to explore whether TGF-@I would act cooperatively with NaB to enhance CE formation. Parallel cultures received vehicle (i.e., acidified PBS) in a volume equal to that received

INTRODUCTION

Cultured normal human epidermal keratinocytes (NHEKs) grown under submerged conditions exhibit ‘To

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AND METHODS

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6467. 27

0014-4827/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

28

WANG

50

40

30

20

10

0

2

1

NaB

3

4

Concentration (mbl)

FIG. 1. Representative example of percentage CEs after 4 days incubation of NHEKs in the presence of 0,0.5, 1.0, or 3.0 n&f NaB. CE formation was correlated with increasing concentrations of NaB. Each data point represents the mean of triplicate samples + SEM.

by treated cultures. Four days later, the number of CEs/lOX microscopic field and the total percentage of detergent-resistant CEs were determined (see below). In a separate series of experiments NHEKs were treated for 4 days with optimal concentrations (3.0 mM) of NaB alone, or 3.0 mi%f NaB + 10 pglml anti-human TGF-P (neutralizing antibodies primarily reactive against human TGF-01) (turkey anti-human TGF-/3 IgG, Collaborative Res., MA). CEs were quantitated and compared to control NHEKs. CE quantitation. Detergent-resistant CEs were quantitated by the method of Gilifix and Green as previously described [ll]. In addition, the number of CEs/lOX field was assessed in situ by direct microscopic examination using paraffin-embedded NHEK sheets which had been detached from the plastic substrate. The embedded cell sheets were sectioned and sequentially stained with xanthene (1 g/ liter) followed by azure A (0.6 g/liter)/methylene blue (0.6 g/liter), and the number of mature, enucleate CEs/lOX field was quantitated as a function of section length.

RESULTS

Exogenous TGF+l Acts Cooperatively with NaB to Induce NHEK Differentiation NaB induces CEs in NHEKs [l] with maximal CE induction occurring at concentrations of 2.5-3.0 mM. While 0.5 and 1.0 mM induced CEs, they did so at considerably lower levels compared to 3 mM NaB (Fig. 1). Matsumoto et al. recently demonstrated that TGF-fi can increase involucrin and CE expression in NHEKs grown in the presence of high Ca2+ [6]. Since NaB increases TGF-fi mRNA abundance, it was possible that TGF-b might influence CE formation in NHEKs which had been treated with suboptimal, low concentrations of

ET AL.

NaB (i.e., 0.5 or 1.0 mA4) in our standard culture medium which contains 1.2 mM Ca2+. NHEKs were coincubated with a combination of suboptimal NaB (0.5 or 1.0 mM) in the presence or absence of TGF+l (1-2 rig/ml) and the prevalence of CEs was compared to control NHEKs (Table 1). Approximately 11 mature, enucleated CE/lOX field was generally seen in control NHEK cell sheets. TGFfil (2 rig/ml) treatment did not up-regulate the expression of CEs/lOX field whereas 3.0 n-J4 NaB induced a lo-fold increase in the number of mature CEs covering the surface of the NHEK cell sheets (6.8 -t- 2.7 CEs/lOX field). Suboptimal, 1.0 mM NaB induced only a moderate increase in CEs (2.9 f 1.3 CE/lOx field). By contrast, combined TGF-61 + 1.0 miVf NaB induced a dramatic increase in CEs/lOX field (8.7 f 2.1), which exceeded the response induced by optimal NaB (Table 1). Quantitation of CEs by detergent resistance gave similar results. TGF-/31 acted cooperatively with suboptima1 concentrations (0.5 or 1.0 mM) of NaB to induce detergent-resistant CEs (54 and 61.5%, respectively) which were equivalent to, or higher in number than, optimal (3.0 n-&f) NaB. Interestingly, however, TGF-/31treated NHEKs revealed higher numbers of CEs by detergent resistance compared to quantitation by microscopy. This discrepancy may be due in part to the technical differences in the assays. Microscopic evaluation was based solely on quantitation of mature enucleate CEs. It is quite possible that a proportion of TGF-/31-treated NHEKs exhibit the detergent resistance typical of mature CEs but are not fully differentiated to the enucleate state. Such immature CEs would not be observed by microscopic evaluation, but may exhibit a detergent-resistant phenotype.

TABLE TGF-01

1

and NaB Act Cooperatively CE Formation in NHEKs

Treatment Control TGF-Bl NaB (0.5 mM) NaB (1.0 n&f) NaB (3.0 m&f) NaB (0.5 m&f) + TGF-01 NaB (1 n&f) + TGF-fll

Percentage CEs f SEM” 11.0 21.4 33.0 42.0 51.5 54.0 61.5

+ + + + + + f

0.6 1.8 4.4 1.4 2.4 2.2d 1.7d

n Results are based on quantitation of detergent [ll]; see also Materials and Methods). * Results are based on microscopic examination enucleate CEs of 10 separate fields per treatment three experiments (for details see Materials and c TGF-/31 concentration, 2 rig/ml. d TGF-01 concentration, 1 rig/ml.

to Induce

Number CEs per 10X field f SD* 0.6 + 0.5 0.4 +- 0.5’ N.D. 2.9 + 1.3 6.8 k 2.7 N.D. 8.7 + 2.1’ resistant

CEs (Ref.

and quantitation of condition in each of Methods).

TGF-fl

TABLE Anti-human

TGF-6

Antibodies

IN NHK

2 Inhibit

the Response

of

NHEKs to NaB Treatment

conditions

Control NaB (3.0 r&f) NaB (3.0 m&f) + anti-human TGF-6 (10 pg/ml)

Percentage CEs (mean -t SEM)” 15.4 + 3.4

50.7 + 2.9 23.7 + 2.2

Note. NaB vs control, P < 0.005. NaB vs NaBlanti-TGF-@, sm. DBased on three sets of experiments conducted in replicate.

Inhibition of Endogenous TGF-(31 Activity NUB-induction of NHEK Differentiation

29

DIFFERENTIATION

P c

Inhibits

Cultured NHEKs secrete TGF-/3 into the culture medium [12] and, when assayed in a growth state appropriate for NaB treatment, secreted -2.3 ng TGF-01 per 10’ cells. To determine whether TGF-/31 played a direct role in CE expression under optimal NaB induction culture conditions, NHEKs were incubated in the presence of either NaB (3.0 mM) alone or a combination of NaB and neutralizing anti-human TGF-P IgG for 4 days, after which time the number of CEs was determined (Table 2). Incubation of NHEKs in 3 mM NaB induced high numbers of CEs compared to control NHEKs (51 vs 15%; P < 0.005). By contrast, addition of anti-TGF-/3 to NaB-treated NHEKs significantly inhibited the NaB-induced differentiation response (51 vs 24% for NaB and NaBlanti-TGF-8, respectively; P -C 0.005). DISCUSSION TGF$s are members of a family of polypeptides which exhibit pleiotropic effects in both epithelial and nonepithelial cell systems [13]. In normal epidermis, TGF-/3 is observed only in the suprabasal cell layers [8] which, in situ, consist of keratinocytes committed to terminal differentiation. Such cell type-specific expression of TGF-/3 strongly suggests that this factor plays a role in maintenance of the differentiated phenotype of NHEKs [8]. While TGF-/3 induces differentiation of bronchial epithelium [14], its effects on NHEK differentiation are less well understood [5-81. TGF$l inhibits cell cycle progression of NHEKs presumably via suppressing phosphorylation of retinoblastoma protein or by downregulation of myc expression [15-181. TGF-/!?l and TGF-@2 appear to foster a wound healing pathway of epidermal differentiation based on keratin expression in NHEKs grown on feeder layers or on floating collagen rafts [5,7]. Prolonged exposure to TGF-Pl or TGF82 may induce differentiation even within basal layer

NHEKs [5]. TGF-j3 may influence NHEK differentiation pathways differently depending upon Ca2+ concentrations. Enhanced CE formation and involucrin expression occurred under high Ca2’ conditions, while differentiation was inhibited under low Ca2’ conditions [6]. However, the level of differentiation achieved was low even after addition of relatively high levels of TGF/31. In the epidermis, terminal differentiation is associated with an increased Ca2+ flux in suprabasal NHEKs which in turn activates transglutaminase, acritical element in CE formation [19]. It is therefore perhaps not surprising that TGF-/? might induce differentiation in NHEKs under high Ca2+ conditions which would be physiologically relevant to epidermis in situ. In the present study, treatment with TGF-Pl alone (l-2 rig/ml) did not increase the number of mature CEs covering the surface of the keratinocyte sheets, but it acted cooperatively with suboptimal NaB levels (0.5 or 1.0 mM) to induce significant expression of mature CEs. Indeed, the combined addition of suboptimal NaB + TGF-/31 generates numbers of CEs which exceed those observed for optimal NaB concentrations. It is not yet clear whether TGF-Pl acts on the same or a unique subpopulation of suprabasal NHEKs and this question is presently under study. The data presented here directly implicate TGF-fil as being necessary for NaB-induced CE formation in NHEKs. Neutralization of TGF-/3 activity with anti-human TGF-P antibodies significantly inhibited CE formation in NaB-treated NHEKs. On the basis of these studies and NaB’s antagonism of retinoid effects it is tempting to speculate that TGF-8 does play a role in the normal cascade of events which culminate in the differentiation of NHEKs. Protein mapping and gene expression studies are underway to define how NaB and TGFfi contribute to CE formation in NHEKs. These studies were supported in part by PHS Grants GM42461 and GM26145 and American Cancer Society Grant SIG-‘IA. We also acknowledge Ms. Bettye Mayer for her excellent assistance in the preparation of this manuscript.

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