Maintenance of the intestinal epithelial barrier is a critical function of small intestinal enterocytes. Activation of the Na+
-glucose cotransporter, SGLT1, initiates a signaling cascade that results in MLC phosphorylation, actomyosin contraction, and increased tight junction permeability, i.e.
reduced barrier function (1
). The latter has been proposed to allow paracellular nutrient absorption, thereby increasing the total absorption rate beyond the saturation point of transcellular transporters when luminal nutrient loads are high (5
). MLCK has been implicated in the control of MLC phosphorylation in this process (1
). Although other kinases and MLC phosphatase can also regulate MLC phosphorylation, the data presented in this study suggest that MLCK plays the principal role in Na+
-nutrient cotransport-dependent tight junction regulation.
Two major isoforms of smooth muscle MLCK exist: short MLCK, found in smooth muscle, and long MLCK, first described in endothelium (14
). In this study we show that long MLCK accounts for the majority of MLC kinase activity in intestinal epithelia. Long MLCK is expressed as multiple splice variants; among these MLCK2 predominates, whereas the full-length transcript, MLCK1, is a minor form (15
). The transcripts for these isoforms differ by only 207 base pairs, but insertion of this 69-amino acid domain can increase MLCK1 activity after phosphorylation by Src family kinases (16
). This 69-amino acid region also contains a potential SH2-binding domain, raising the possibility that there are protein interactions unique to MLCK1. Previous studies have confirmed that MLCK1 and MLCK2 are coexpressed in a variety of tissues but did not identify differences in expression pattern that could suggest unique roles for each splice variant (15
Given that multiple MLCK-dependent processes, including Na+
-glucose cotransport-dependent barrier regulation and extrusion of apoptotic enterocytes, are precisely localized along the crypt-villus axis, we hypothesized that MLCK isoform expression might parallel epithelial function. We took advantage of the ordered system of enterocyte differentiation to assess MLCK isoform expression, both in the in vitro
Caco-2 cell model of enterocyte differentiation (21
) and in vivo
, as assessed in human intestine. Analysis of mRNA from Caco-2 cells and human intestinal epithelia showed that long MLCK expression was limited to isoforms 1 and 2. Semiquantitative RT-PCR showed that MLCK1 expression increased progressively during enterocyte differentiation, both in vitro
and in vivo
, and immunofluorescence microscopy confirmed that mRNA increases are matched by increased villus expression of MLCK1.
Our data show that MLCK1 expression is low in undifferentiated, proliferating (crypt) enterocytes but increases markedly during differentiation into absorptive (villus) enterocytes. MLCK1 mRNA expression was regulated similarly in vitro
and in vivo
: Caco-2 cells demonstrate an increase in MLCK1 levels at 6 days postconfluence, whereas human jejunal enterocytes show increased MLCK1 mRNA at the villus tip, 3–5 days after cells exit the crypt compartment (31
). This increased MLCK1 expression strongly correlated with Na+
-glucose cotransport-dependent tight junction regulation, which is limited to the villus in native intestine (4
) and, as we show here, occurs only in differentiated Caco-2 monolayers ≥6 days post-confluence.
In addition to restriction of MLCK1 expression to well differentiated absorptive enterocytes, MLCK1 demonstrated a unique subcellular localization that suggested functional specialization of this isoform. MLCK1 is restricted to the apical enterocyte cytoplasm in the area of the perijunctional actomyosin ring. Both this crypt to villus distribution and subcellular localization are similar to those described here and previously described (20
), respectively, for phosphorylated MLC. Thus, MLCK1 may be responsible for perijunctional MLC phosphorylation in villus enterocytes.
To directly test the role of MLCK1 in tight junction regulation, we selectively knocked down MLCK1 using isoform-specific siRNA. MLCK1 knockdown increased TER, i.e.
decreased tight junction permeability, in monolayers with active Na+
-glucose cotransport. This result confirms that the localization of MLCK1 to the perijunctional actomyosin ring is functionally significant and explains the strong correlation between development of physiological tight junction regulation and expression of MLCK1 during enterocyte differentiation. Thus, we must ask how a small 69-amino acid insert in a 1914-amino acid protein can result in such specialized localization and function. The answer may lay in definition of the binding partners and enzymatic regulators of motifs within this region. Given that MLCK-mediated barrier dysfunction has been implicated in diverse disease processes (12
), definition of these regulatory motifs within MLCK1 may both enhance our understanding of physiology and provide novel therapeutic targets for a variety of disorders.
In summary, the data show that the MLCK expressed in intestinal epithelium is long MLCK and that the two isoforms expressed, MLCK1 and MLCK2, exhibit precisely regulated expression along the crypt-villus axis. The expression of MLCK1 is restricted to absorptive villus enterocytes, and increased levels of this isoform correlate strongly with the development of physiologic tight junction regulation. Furthermore, MLCK1 is localized to the perijunctional actomyosin ring at the level of the tight junction. Knockdown of MLCK1 leads to decreased tight junction permeability, demonstrating that MLCK1 controls tight junction regulation in small intestinal enterocytes. Thus, the short sequence that distinguishes MLCK1 from MLCK2 endows this isoform with a unique functional role in intestinal epithelium