Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which β-actin is also regulated, the mRNA for β-actin remains a widely used housekeeping gene internal control. Utilizing differential reverse transcriptase-polymerase chain reaction (RT-PCR), we report here the dose-dependent inhibition of β-actin by matrigel. This was detected by comparison to the very moderate inhibition of the target gene, membrane type-1 matrix metalloproteinase (MT1-MMP), with results independently confirmed by similar findings on MT1-MMP expression using competitive RT-PCR. Furthermore, RT-PCR of the housekeeping gene 18 Svedberg Units (S) rRNA demonstrated excellent consistency, reproducibility and non-regulation by a matrigel treatment. We conclude that β-actin is highly regulated by matrigel and therefore unsuitable as an internal control in this treatment. Hence, these findings suggest that researchers have a responsibility to ensure that the housekeeping gene of choice is not regulated in their specific application, as such regulation may dramatically affect the accuracy of their results. This study reinforces the necessity for minimally regulated housekeeping genes such as 18S rRNA, and the superiority of competitive templates as internal controls for quantitative applications of RT-PCR.

β-Actin—an unsuitable internal control for RT-PCR

Stromelysin-2 is a matrix metalloproteinase that degrades in vitro several protein components relevant to wound repair such as collagens III and IV, gelatin, nidogen, laminin-1, proteoglycans, and elastin. Furthermore, it can activate other matrix metalloproteinases, such as collagenase-1 (matrix metalloproteinase-1) and collagenase-2 (matrix metalloproteinase-8), as well as 92 kDa gelatinase. The aim of this study was to determine in a large variety of wounds (normally healing dermal and mucosal wounds, suction blisters, ex vivo cultures, diabetic, decubitus, rheumatic, and venous ulcers) and keratinocyte cultures, which factors contribute to stromelysin-2 expression and how it is induced in relation to other matrix metalloproteinases. Our results show that stromelysin-2 mRNA and protein are upregulated later (at 3 d) than matrix metalloproteinase-1 in normally healing wounds and ex vivo explants, in which stromelysin-2 is invariably expressed by keratinocytes migrating over dermal matrix. The number of keratinocytes expressing stromelysin-2 was greatest in chronic inflamed diabetic and venous ulcers compared with rheumatoid and decubitus ulcers, six of which had no signal. In keratinocyte cultures, tumor necrosis factor-α, epidermal growth factor, and transforming growth factor-β1 induced stromelysin-2 expression as measured by quantitative reverse transcriptase–polymerase chain reaction, whereas different matrices did not upregulate the mRNA. Immunostaining demonstrated stromal transforming growth factor-β1 in contact with the stromelysin-2-positive keratinocytes. Our results suggest that stromelysin-2 expression is important for the normal repair process and is upregulated by cytokines rather than cell–matrix interactions. Stromelysin-2 is most likely to participate in the remodeling of the newly formed basement membrane, and is not overexpressed in retarded wound healing.

Stromelysin-2 is upregulated during normal wound repair and is induced by cytokines

Epithelial cells provide barrier protection throughout the body. Although oral mucosal and skin epithelium share many morphological similarities they also exhibit distinct capacities.
One significant functional difference is the wound healing response, as mucosal wounds heal more rapidly than skin (Szpaderska 2005). The structure of the oral mucosa resembles the skin in many ways. It is composed of two layers, the epithelium and connective tissue. The epithelium of the oral mucosal membrane may be keratinized or nonkeratinized depending on location. In humans and mice, the dorsum of the tongue, gingiva and hard palate are keratinized tissues. Keratinized tissue in the oral mucosa is very similar to skin, making it a good model to study healing.
One important aspect of wound healing is re-epithelialization, which is restoration of the epidermis by keratinocytes. Upon injury, epithelial cells from wound edges dissolve their hemidesmosomal connections, detach from the basement membrane and move quickly across the exposed connective tissue. In the normal situation, cells just behind the wound edge undergo a proliferative and migratory burst and effectively replace keratinocytes lost as a result of injury. This tremendous proliferative and migratory capacity of keratinocytes is critical for effective wound re-epithelialization.
In a study where equivalent full thickness 1mm diameter wounds are created on both the dorsal skin and tongue of the mouse, oral mucosa wounds
ix
exhibit re-epithelialization very rapidly, with 100% completion at 24 hours post-injury. In contrast, cutaneous wounds are less than 25% re-epithelialized at the 24 hour time point (Schrementi 2008). These data strongly suggest that the proliferative capacity of oral keratinocytes is greater than that of dermal keratinocytes. As migration is another critical aspect of re-epithelialization, in this study we investigate whether the migratory capacity of oral keratinocytes is different in comparison to skin.
Evidence to support the hypothesis that differences responsible for rapid repair in oral mucosa could be intrinsic keratinocyte characteristics is available. Since keratinocytes are the major source of VEGF in the wound and produce pro-angiogenic factors in response to hypoxia, investigation of the VEGF mRNA and protein levels in isolated oral and skin keratinocytes was conducted. When subjected to hypoxia, epidermal keratinocytes produced higher levels of both VEGF protein and mRNA than did oral keratinocytes (Szpaderska 2005). These findings imply that the superior repair in oral mucosa is likely to be related to intrinsic characteristics of mucosal tissue and not to environmental factors such as temperature, salivary flow or microflora.
The goal of this study was to identify innate positional differences in epithelial cells that might contribute to the site-specific response to injury.

Differences Between Oral and Skin Keratinocytes During Wound Healing

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Differences Between Oral and Skin Keratinocytes During Wound Healing