HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/10/1352 02-0959fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MACK, J. A. Articles by STELNICKI, E. J. Search for Related Content PubMed PubMed Citation Articles by MACK, J. A. Articles by STELNICKI, E. J.

Hoxb13 knockout adult skin exhibits high levels of hyaluronan and enhanced wound healing¹

JUDITH A. MACK^*,,,2, SUSAN R. ABRAMSON^*,, YIXEN BEN^*, JERUSHA C. COFFIN^*, JAMES K. ROTHROCK^*, EDWARD V. MAYTIN^,, VINCENT C. HASCALL, COREY LARGMAN^¶ and ERIC J. STELNICKI

^* Department of Research, Cleveland Clinic Florida, Weston, Florida, USA;
College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Florida, USA;
Department of Biomedical Engineering, Cleveland Clinic Foundation, Cleveland, Ohio, USA;
Department of Dermatology, Cleveland Clinic Foundation, Cleveland, Ohio, USA; and
^¶ Departments of Dermatology and Medicine, VA Medical Center and University of California, San Francisco, California, USA

²Correspondence: Cleveland Clinic Foundation, Lerner Research Institute, Department of Biomedical Engineering/ND20, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: mackj{at}ccf.org

SPECIFIC AIMS

Expression levels of the transcriptional regulator HoxB13 are significantly reduced in human fetal cutaneous wounds that heal without a scar compared with unwounded controls. In contrast, HoxB13 down-regulation is not noted in human adult cutaneous wounds vs. unwounded controls. We postulated that reducing or eliminating HoxB13 from adult skin could improve healing. To test this hypothesis, we evaluated incisional and excisional cutaneous wound healing in adult Hoxb13 knockout (KO) mice.

PRINCIPAL FINDINGS

1. Hoxb13 is expressed in murine skin
Hoxb13 mRNA is present in the epidermal and dermal layers of the skin and in RNA derived from primary keratinocyte and fibroblast cell cultures. Hoxb13 protein is detected in the nucleus and cytoplasm of the epidermis, epithelial cells of the hair follicle, and dermal fibroblasts and is up-regulated at the wound site. These observations indicate a functional role for Hoxb13 in the epidermal and dermal layers of the skin.

2. Hoxb13 KO adult skin exhibits a thick dermal layer and high levels of hyaluronan (HA)
Hoxb13 KO mice are homozygous viable with no apparent skin abnormalities, although the average thickness of Hoxb13 KO dermis (346±150 µm) (Fig. 1 B) was significantly greater than that of WT dermis (236±81 µm; P<0.02; Student’s t test) (Fig. 1A ). Hydroxyproline analysis indicated the difference was not due to higher amounts of collagen in Hoxb13 KO dermis than WT dermis (154±9 µg/mg dry weight vs. 155±52 µg/mg dry weight, respectively). We then asked whether a higher HA content in Hoxb13 KO skin could account for the thicker dermal phenotype by increasing the extracellular volume of the dermis. Adult full-thickness skin sections stained with a biotinylated hyaluronan binding protein (bHABP) showed significantly higher levels of HA in Hoxb13 KO epidermis (Fig. 1D ) than WT epidermis (Fig. 1C ). Since staining intensity was very high in both Hoxb13 KO and WT dermis, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to analyze HA content in the dermis. Densitometry analysis showed a twofold increase in HA levels (white arrow) in Hoxb13 KO dermis compared with WT dermis (Fig. 1E ).

View larger version (92K): [in this window] [in a new window]   Figure 1. Hematoxylin and eosin stained full-thickness sections from WT (A) and Hoxb13 KO (B) adult skin. Black arrows span the dermis. The red arrow spans the fat layer. bHABP stained full-thickness skin sections from WT (C) and Hoxb13 KO (D) adult skin. White dashed lines outline the epidermis. E) FACE analysis. Upper bands (white arrow) consist of HA disaccharides derived from skin from two WT adult mice (1, 2) and two Hoxb13 KO mice (3, 4) after digestion with hyaluronidase. Lower bands consist of disaccharides of chondroitin sulfate/dermatan sulfate (CS/DS) from the same animals as above obtained after digestion with Chondroitinase ABC. Densitometry analysis showed there was no difference in CS/DS levels between Hoxb13 KO and WT dermis. Normalizing HA with CS/DS indicated a twofold increase in HA levels in Hoxb13 KO dermis.

3. Hoxb13 KO excisional wounds close faster than WT excisional wounds
HA has been implicated as an important factor in fetal scarless wound healing. To assess healing in Hoxb13 KO mice, we performed full-thickness 6 mm diameter excisional wounds on the upper back of Hoxb13 KO and WT mice (8–16 wk) (n=10 each for WT and Hoxb13 KO) and determined the rate of closure over several time points. Overall, the closure rate of Hoxb13 KO wounds was significantly greater than WT wounds (P<0.05, ANOVA). The most striking difference was observed 24 h after wounding where the average Hoxb13 KO wound was > 20% smaller than the average WT wound (P<0.01, Student’s t test).

4. Hoxb13 KO incisional wounds exhibit enhanced healing
To further assess healing, we made full-thickness incisional wounds on the upper back of Hoxb13 KO and WT adult mice (8–16 wk) (n=10 each for WT and Hoxb13 KO at each time point). Wounds were harvested at 3, 5, 7, 14, 21, 30, and 60 days postwounding for histological and mechanical testing (tensiometry). Evaluation of hematoxylin and eosin stained sections of skin harvested 7 days after wounding showed that the average size of the WT wound (0.23±0.16 mm², n=8) (Fig. 2 A) was more than twice the size of the average Hoxb13 KO wound (0.097±00.072 mm², n=9, P<0.05, Student’s t test) (Fig. 2B ). Tensiometry analysis demonstrated that the overall breaking strength of a Hoxb13 KO wound was significantly greater than that of a WT wound (P<0.05, ANOVA). At day 60 postwounding, the average tensile strength of a Hoxb13 KO wound was comparable to than of Hoxb13 KO unwounded skin and significantly greater that the average WT 60-day-old wound (P<0.02, Student’s t test).

View larger version (134K): [in this window] [in a new window]   Figure 2. Hematoxylin and eosin stained full-thickness sections from WT (A) and Hoxb13 KO (B) skin harvested 7 days after incisional wounding. The wounded region is in the center of the section and encircled in black. Arrows span the unwounded dermis on either side of the wound. Mason’s Trichrome stained full thickness sections containing 60-day-old incisional wounded/remodeled tissue from WT (C') and Hoxb13 KO (D') skin and adjacent unwounded WT and Hoxb13 KO tissue (C and D, respectively). Collagen stains in a blue-green color.

5. Collagen is reconstituted in a more normal pattern in Hoxb13 KO wounds
The tensile strength of healed wounds is determined primarily by how well the collagen has remodeled. To evaluate remodeling, we stained full-thickness skin sections containing 60-day-old incisional wound tissue plus adjacent unwounded tissue with Masson’s trichrome for collagen. In unwounded WT and Hoxb13 KO dermis (Fig. 2C, D , respectively), the collagen is loose and reticulate. In the WT wound (Fig. 2C '), the collagen is arranged in dense parallel bundles typical of adult scar tissue. In the Hoxb13 KO wound (Fig. 2D '), the collagen aggregation is looser and more interwoven, resembling that of unwounded skin, indicating that collagen remodeling in Hoxb13 KO wounds is producing a more normal dermal architecture.

CONCLUSION AND SIGNIFICANCE

Inadequate wound healing and/or scarring can result in major clinical problems affecting structure, function, and quality of life. Therefore, any potential avenues leading to improved healing and reduced scarring would be of great benefit. Here we demonstrate that loss-of-Hoxb13 function in adult mouse skin results in enhanced cutaneous wound healing and higher levels of HA in both the epidermal and dermal layers of the skin.

In normal skin, HA levels are significantly higher in fetal skin and fetal cutaneous wounds than in adult skin/wounds. HA is thought to promote cell migration and proliferation and inhibit differentiation. Because of these characteristics, HA has been implicated as an important factor in fetal scarless wound healing. In addition to a scarless phenotype, fetal cutaneous wounds are characterized by faster closure compared with adult wounds. During the first 1 to 2 days after injury, wound closure is accomplished by epidermal cell migration after which the cells begin to proliferate. The most significant result comparing epidermal closure rates between Hoxb13 KO and WT excisional wounds occurred in the first 24 h, at which time the average Hoxb13 KO wound size was >20% smaller than the average WT wound size. We hypothesize that Hoxb13 KO epidermis, already enriched in HA at the time of wounding, provides an environment immediately favorable for early and rapid keratinocyte migration (Fig. 3 ). Support for this comes from studies by others. HA is synthesized by one or more hyaluronan synthases (HAS1, HAS2, and/or HAS3). Rat 3Y1 fibroblasts accumulating abnormally high levels of HA as a result of HAS2 overexpression demonstrated enhanced motility in scratch wound assays. Conversely, reduced migration was noted in a rat keratinocyte cell line overexpressing antisense HAS2 with concurrent loss of HA.

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the potential effects of high levels of HA in Hoxb13 KO skin during early and late wound healing. The relatively high levels of HA present in Hoxb13 KO epidermis compared with WT epidermis at the time of excisional wounding results in a significantly faster rate of keratinocyte migration at the Hoxb13 KO wound edge during the first 24 h postwounding. The relatively high levels of HA present in Hoxb13 KO dermis during the later stages of wound healing compared with WT dermis favors the restoration of a more normal collagen architecture in Hoxb13 KO remodeling wounds.

Reconstitution of prewound collagen (dermal) architecture is a hallmark of fetal scarless wound healing. During normal skin repair, the complex process of collagen remodeling begins 3 wk after injury and continues until ca. day 100 postwounding. The success of collagen remodeling is a primary determinant of tensile strength in healed wounds. Generally, adult healed cutaneous wounds attain only 70–80% of the tensile strength of unwounded skin, which we found to be the case when comparing the average tensile strength of 60-day-old WT incisional wounds with WT unwounded skin. In sharp contrast, the average tensile strength of 60-day-old Hoxb13 KO incisional wounds was comparable to that of unwounded Hoxb13 KO skin. We believe this can be explained by our observation that in Hoxb13 KO day 60 wounds, collagen is organized in a more normal reticular pattern as opposed to the scarred pattern of WT wounds.

It has been suggested that a high and persistent HA concentration in the fetal wound may influence the nature of the collagen fibrils formed, resulting in a prewound pattern of collagen deposition. This hypothesis is supported by previous studies conducted by others. In one such study, HA-treated injured menisci showed a greater level of collagen remodeling compared with untreated injured menisci. In another study, treatment of synovium with hyaluronidase, an enzyme that degrades HA, differentially influenced collagen fibril formation, indicating that HA chains have a major organizational role within collagen bundles. Based on these observations, we speculate that attainment of a more normal dermal architecture in Hoxb13 KO wounds is due in part to the persistently higher levels of HA present in Hoxb13 KO adult dermis throughout the remodeling process (Fig. 3) .

HA is normally synthesized by keratinocytes and fibroblasts. Our results argue that one function of Hoxb13 is to down-regulate HA production in these cells. To accomplish this, Hoxb13 could directly suppress the expression of one or more of the HAS genes or, conversely, directly activate the expression of one or more of the hyaluronidase genes. Alternatively, Hoxb13 may influence HA levels indirectly by regulating the expression of genes upstream of the HA metabolic pathways. Based on the higher levels of HA in Hoxb13 KO adult skin, we speculate that one role for Hoxb13 in adult skin is to promote differentiation during epidermal renewal by keeping levels of HA low. This is supported by our initial array analyses indicating that the expression levels of several epidermal differentiation markers are reduced in adult Hoxb13 KO skin. If Hoxb13 does indeed play a role in promoting epidermal differentiation, then there are other molecules involved in this process, as loss of Hoxb13 alone does not appear to affect the overall integrity or health of the skin. However, our results suggest that eliminating Hoxb13 in adult skin changes its differentiation status in such a way as to promote enhanced wound healing. The fact that eliminating Hoxb13 does not appear to harm skin while improving wound repair identifies Hoxb13 as an ideal clinical target in situations of impaired healing.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0959fje; doi: 10.1096/fj.02-0959fje

This article has been cited by other articles:

				V. Muthusamy, S. Duraisamy, C. M. Bradbury, C. Hobbs, D. P. Curley, B. Nelson, and M. Bosenberg Epigenetic Silencing of Novel Tumor Suppressors in Malignant Melanoma Cancer Res., December 1, 2006; 66(23): 11187 - 11193. [Abstract] [Full Text] [PDF]

				J. Monslow, J. D. Williams, D. J. Fraser, D. R. Michael, P. Foka, A. P. Kift-Morgan, D. D. Luo, C. A. Fielding, K. J. Craig, N. Topley, et al. Sp1 and Sp3 Mediate Constitutive Transcription of the Human Hyaluronan Synthase 2 Gene J. Biol. Chem., June 30, 2006; 281(26): 18043 - 18050. [Abstract] [Full Text] [PDF]

				A.A. DeCarlo and J.M. Whitelock The Role of Heparan Sulfate and Perlecan in Bone-regenerative Procedures J. Dent. Res., February 1, 2006; 85(2): 122 - 132. [Abstract] [Full Text] [PDF]

				J. A. Mack, L. Li, N. Sato, V. C. Hascall, and E. V. Maytin Hoxb13 Up-regulates Transglutaminase Activity and Drives Terminal Differentiation in an Epidermal Organotypic Model J. Biol. Chem., August 19, 2005; 280(33): 29904 - 29911. [Abstract] [Full Text] [PDF]

				E. V. Maytin, H. H. Chung, and V. M. Seetharaman Hyaluronan Participates in the Epidermal Response to Disruption of the Permeability Barrier in Vivo Am. J. Pathol., October 1, 2004; 165(4): 1331 - 1341. [Abstract] [Full Text] [PDF]

				J. Monslow, J. D. Williams, C. A. Guy, I. K. Price, K. J. Craig, H. J. Williams, N. M. Williams, J. Martin, S. L. Coleman, N. Topley, et al. Identification and Analysis of the Promoter Region of the Human Hyaluronan Synthase 2 Gene J. Biol. Chem., May 14, 2004; 279(20): 20576 - 20581. [Abstract] [Full Text] [PDF]

				C. Jung, R.-S. Kim, S.-J. Lee, C. Wang, and M.-H. Jeng HOXB13 Homeodomain Protein Suppresses the Growth of Prostate Cancer Cells by the Negative Regulation of T-Cell Factor 4 Cancer Res., May 1, 2004; 64(9): 3046 - 3051. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/10/1352 02-0959fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MACK, J. A. Articles by STELNICKI, E. J. Search for Related Content PubMed PubMed Citation Articles by MACK, J. A. Articles by STELNICKI, E. J.